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EFFECTS OF PAPER MILL EFFLUENTS ON THE HEALTH AND 
REPRODUCTIVE SUCCESS OF LARGEMOUTH BASS (MICROPTERUS 
SALMOIDES): FIELD AND LABORATORY STUDIES 



By 
MARIA SOLED AD SEPULVEDA 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

2000 



ACKNOWLEDGMENTS 

My deepest thanks to Dr. Timothy Gross for having accepted me as his graduate 
student. He introduced me to the field of fish ecotoxicology and endocrine disruption, 
and offered me the unique opportunity to work with an interdisciplinary research team at 
the USGS-BRD Florida Caribbean Science Center Ecotoxicology Laboratory 
(Gainesville, FL). Ecotoxicology staff members and friends Shane Ruessler, Carla 
Wieser, Jon Wiebe, Nikki Kernaghan, Kelly McDonald, and Vincent and Lisa Centonze 
assisted in innumerable tasks ranging from the treatment, collection and processing of 
fish; analysis of plasma samples for reproductive hormones; to the conduction of 
spawning studies. Lyn Day and Meri Nantz helped in the purchase of equipment and in 
the day-to-day process of trying to get things done. My most sincere appreciation to all of 
them. I will always remember my days at the "ecotoxicology lab"! 

I would also like to thank my other committee members: Drs. Evan Gallagher and 
Steve Roberts (Center for Environmental and Human Toxicology, College of Veterinary 
Medicine, UF, Gainesville, FL); Dr. Trenton Schoeb (Department of Pathobiology, 
College of Veterinary Medicine, UF, Gainesville, FL), and Dr. Nancy Denslow 
(Department of Biochemistry and Molecular Biology, College of Medicine, UF, 
Gainesville, FL). Their comments and suggestions greatly improved the quality of my 
research and writing. Also, Dr. Schoeb's help in the interpretation of histology slides and 



11 









Dr. Gallagher's assistance in the conduction of EROD and other liver enzyme assays are 
greatly acknowledged. 

This dissertation would not have been possible without the support of the 
Georgia-Pacific Corporation, Atlanta, GA. through a two-year grant awarded to Dr. 
Timothy Gross. My deepest thanks to Stewart Holm, from Georgia-Pacific, for his help 
in designing and overseeing this project. Also thanks to Myra Carpenter (Georgia- 
Pacific, Palatka Operation, FL) who was responsible for the building and maintenance of 
the treatment tank system in Palatka. 

I also thank Karen Sheehy (Center for Environmental and Human Toxicology, 
UF, Gainesville, FL) for her assistance in the EROD analyses, as well as Kevin Kroll and 
Marjorie Chow (Center for Biotechnology, UF, Gainesville, FL) for conducting the 1998 
vitellogenin analyses. I am specially thankful to Kevin, who kindly spent time training 
me in the "art" of conducting ELISAs for the detection of vitellogenin in bass. 

I would like to thank Bill Johnson and other staff from the Florida Game and 
Freshwater Fish Commission (Fisheries Research Laboratory, Eustis, FL) for providing 
boats and personnel for the collection of largemouth bass from the St. Johns River and for 
aging the fish using otolith analyses. John Higman (St. Johns Water Management 
District) provided invaluable information on fish and sediment chemical data from sites 
along the lower St. Johns River. Jay Harrison and Galin Jones (Department of Statistics, 
UF, Gainesville, FL) assisted in the statistical analyses. 

Finally, with special recognition and love, I thank my husband, Hugo Ochoa, my 
daughter Natalia Ochoa, and my mother Pura Luque, for their invaluable collaboration, 



111 






support, and most of all for their patience. I would not have been able to finish this 
degree without them. 



iv 






TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS ii 

ABSTRACT viii 

CHAPTERS 

1 INTRODUCTION AND BACKGROUND 1 

Introduction 1 

General Aims of Ecotoxicological Studies 2 

Biomarkers of Exposure and Effects 2 

Levels of Biological Organization 3 

Effects of Endocrine Disrupting Chemicals in Wildlife 4 

The Pulp and Paper Industry 5 

Introduction 5 

Pulp and Paper Manufacturing Process Sequence 5 

Pollution Outputs 9 

Wastewater Treatment Technology 10 

The Pulp and Paper Cluster Rules 1 1 

Georgia-Pacific's Paper Mill Plant in Palatka, Florida 12 

General Description 12 

Ongoing Improvements 13 

Sublethal Physiological Effects of Pulp and Paper Mill Effluents on Fish 13 

General Health Effects 14 

Liver Health Effects 17 

Reproductive Health Effects 22 

The Largemouth Bass {Micropterus salmoides) 27 

General Description 27 

Geographic Distribution 28 

Habitat and Range 28 

Growth and Feeding Habits 29 

Reproduction 30 

Significance of this Work 33 

Organization of Dissertation 34 

2 COMPARISON OF REPRODUCTIVE PARAMETERS FROM FLORIDA 
LARGEMOUTH BASS {MICROPTERUS SALMOIDES FLORIDANUS) 
SAMPLED FROM REFERENCE AND CONTAMINATED SITES IN THE 

ST. JOHNS RIVER AND TRIBUTARIES 36 



Introduction 36 

Materials and Methods 39 

Sampling Sites and Fish Collection 39 

Chemical Analysis from Fish Tissues 40 

Bleeding, Necropsies, and Age Determination 41 

Reproductive Endpoints 41 

Liver EROD Activity 46 

Statistical Analyses 47 

Results 48 

Chemical Analysis from Sediments and Fish Tissues 48 

Physiological and Reproductive Endpoints 48 

Discussion 53 

3 IN VIVO ASSESSMENT ON THE REPRODUCTIVE EFFECTS OF 

PAPER MILL EFFLUENTS ON LARGEMOUTH BASS 91 

Introduction 91 

Materials and Methods 92 

Animals and Holding Facility 92 

Effluent Characteristics 93 

Exposure Conditions 94 

Reproductive Endpoints 94 

Statistical Analyses 95 

Results 96 

Females 96 

Males 98 

Both Sexes 99 

Discussion 100 

4 IMPACT OF PAPER MILL EFFLUENTS ON LARGEMOUTH BASS 
HEALTH: FIELD AND LABORATORY STUDIES 128 

Introduction 128 

Materials and Methods 129 

Field Study 129 

Laboratory Study 134 

Results 136 

Field Study 136 

Laboratory Study 137 

Discussion 138 

5 EFFECTS OF PAPER MILL EFFLUENTS ON REPRODUCTIVE 

SUCCESS OF LARGEMOUTH BASS 165 

Introduction 165 

Materials and Methods 166 

In Vivo Experiment 166 

Spawning Study 169 

Results 173 



VI 



In Vivo Experiment 173 

Spawning Study 178 

Discussion 180 

6 IN VITRO STEROIDOGENESIS BY GONADAL TISSUES FROM 
FEMALE LARGEMOUTH BASS EXPOSED TO PAPER MILL 
EFFLUENTS AND RESIN ACIDS 224 

Introduction 224 

Materials and Methods 225 

Effluent Characteristics 225 

In Vivo Exposures 226 

In Vitro Gonadal Cultures 227 

Statistical Analyses 228 

Results 229 

Experiment 1 229 

Experiment 2 229 

Experiment 3 230 

Discussion 230 

7 GENERAL CONCLUSIONS , ECOLOGICAL SIGNIFIC ANCE, AND 
FUTURE RESEARCH NEEDS 242 

General Conclusions 242 

Field Studies 242 

In Vivo Studies 243 

In Vitro Studies 245 

Ecological Significance 245 

Future Research Needs 247 

Additional Field Studies 248 

Mesocosms Studies 248 

Evaluate Effects on Other Aquatic Organisms 249 

Evaluate Biological Effects of Mill Improvements 249 

Mechanistic Studies 250 

REFERENCES 248 

BIOGRAPHICAL SKETCH 268 



VI 1 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 

EFFECTS OF PAPER MILL EFFLUENTS ON THE HEALTH AND REPRODUCTIVE 
SUCCESS OF LARGEMOUTH BASS (MICROPTERUS SALMOIDES): FIELD AND 

LABORATORY STUDIES 

By 

Maria S. Sepulveda 

August 2000 

Chairman: Timothy S. Gross 

Major Department: Veterinary Medicine 

The effects of bleached kraft paper mill effluents (BKME) on the health and 
reproduction of largemouth bass (Micropterus salmoides) were examined through field 
and laboratory studies. During 1996/97 and 1998, bass were collected from both BKME- 
exposed (located at different distances downstream from the effluents discharged by the 
Palatka Paper Mill) and reference streams, and parameters compared across sites. 
Although concentrations of sex steroids and vitellogenin and induction of 
biotransformation enzymes were altered in bass from exposed streams, there were no 
differences on gonad weights, fecundities, and age distributions across sites. Some health 
endpoints were altered in bass collected from exposed streams, but these fell within 
normal ranges and were probably not associated with detrimental health effects. 

Laboratory studies that involved exposures of bass to different concentrations of 
BKME (10, 20, 40, and 80%) for up to 56 days were conducted during the reproductive 



Vlll 



seasons of 1998 and 1999. In contrast to what was observed in the field, bass exposed to 
Palatka's BKME responded with changes at the biochemical-level (decline in sex steroids 
and vitellogenin) that were usually translated into tissue/organ-level responses (declines 
in gonad weights and retardation of gonad development). The majority of these responses 
were observed after exposures to at least 20% BKME concentrations. These changes, 
however, did not result in lower fecundities, egg sizes, or hatchabilities. Later 
evaluations of fry numbers revealed significant negative effects of effluent exposure on 
survivorship, with a threshold effluent concentration of 10%. This decline was probably 
caused by an increased frequency of deformities coupled with alterations on growth. It 
was hypothesized that these changes could have resulted from alterations in "egg quality" 
due to chronic failure of parental reproductive systems after almost two months of 
effluent exposures, and/or to acute embryo toxicity after translocation of persistent 
organic compounds from the mother to the developing embryo. 

Results from in vitro cultures showed significant declines in the production of 70- 
estradiol by follicles collected from BKME-exposed females. These declines paralleled 
changes in plasma 17p-estradiol observed in females during the in vivo studies, and 
suggested the direct action of chemical(s) at the gonad level. There were no dose- 
response changes associated with resin acid exposures, which would suggest the action of 
chemicals other than resin acids as possible causative agents of the reproductive 
alterations observed in BKME-exposed largemouth bass. 



IX 



CHAPTER 1 
INTRODUCTION AND BACKGROUND 



Introduction 



For over a century, men have used seas, lakes, rivers, and other sources of water 
as final resting points for many industrial and agricultural contaminants. This chemical 
release to the environment has prompted scientists around the world to evaluate the 
potential effects of such pollutants on both human and ecosystem health. Since fish play 
a fundamental role in aquatic ecosystems, they have been widely used as monitors of 
environmental health and quality. Earlier studies on the effects of environmental 
contaminants in fish and other wildlife were focused on examining and reporting obvious 
and rather catastrophic responses, such as big kills or die-offs. Because stricter 
environmental regulations nowadays have overall decreased the toxicity of chemicals that 
are being released to the environment, acute lethality is no longer a likely response in 
wild populations inhabiting contaminated areas. Long-term exposure to low 
concentrations of pollutants, however, is still of major concern because it can seriously 
affect the ability of individual animals to grow, survive to adulthood, and reproduce 
leading to population number declines and eventually extinction. Measuring these subtle 
responses in fish populations in response to environmental contaminants is the focus of 
this dissertation. 



The following literature review will first cover information regarding some 
general aspects of ecotoxicological studies; a definition of biomarkers of exposure and 
effects and of levels of biological organization; and a brief summary on the effects of 
endocrine disrupting chemicals in wildlife. Since this dissertation evaluated the potential 
effects of paper mill effluents, a general description of the pulp and paper industry and of 
the sublethal effects of effluent exposure on fish will follow. Biological and life history 
information regarding the study model used in this dissertation (the largemouth bass) is 
presented next. Finally this first chapter ends with a statement on the significance of this 
work and with a description on the way this dissertation was organized. 
General Aims of Ecotoxicological Studies 

Ecotoxicology is an interdisciplinary science that integrates analytical, 
toxicological, and ecological information to predict the fate and adverse effects of 
chemicals on ecosystems (Brouwer et al. 1990). It is important to stress, however, that 
this kind of information can only be obtained by conducting paired "field" and 
"laboratory" studies. Whereas ecological and analytical information is mainly gathered 
from field studies, toxicological data is mostly obtained through controlled laboratory 
studies. Both types of studies are complementary in nature, and if well designed, should 
help decrease the gap between cause and effect relationships and provide useful 
information for developing ecological risk assessment models. 
Biomarkers of Exposure and Effects 

Biomarkers are quantifiable measures of either exposure or effects to 
environmental stresses, such as environmental contaminants. The former indicate that 
exposure to certain chemical (s) has occurred, but gives no information on potential 



effects associated with such an exposure. An example of this type of biomarker would be 
the induction of biotransformation enzymes (e.g., cytochrome P450 monooxygenases) 
after exposure of fish to planar aromatic and halogenated hydrocarbons (Stegeman et al. 
1992). A biomarker of effect, on the other hand, usually measures biochemical, 
physiological, or histological adverse changes that result after exposure to certain 
chemicals, for example, an increase in fry malformations after exposure of fish to 
persistent compounds such as dioxins (Henry et al. 1997). The major goals of the 
biomarker approach are to evaluate sublethal effects of chemicals; predict future trends 
(i.e. serve as early warning indicators); monitor the distribution, changes, and persistence 
of environmental pollutants; and whenever possible, establish cause and effects 
relationships (Adams et al. 1989). 
Levels of Biological Organization 

Exposure to environmental chemicals, however, usually leads to changes at 
different levels of biological organization (i.e. molecule, organelle, cell, tissue, organ, 
individual, population, community, and ecosystem). This means that when trying to 
evaluate the effects of environmental contaminants on fish, a variety of responses at 
several organizational levels are needed if biological and ecological meaningful results 
are intended. In other words, no single method or index can provide all the necessary 
information to understand the condition of a fish population or community. Indicators 
that reflect conditions at lower organizational levels (such as molecular and biochemical 
levels) respond relatively rapidly to stress and have high toxicological relevance; on the 
other hand, indicators that reflect conditions at higher organizational levels (such as 



organism and population-levels) respond more slowly and have less toxicological but 
more ecological relevance (Adams et al. 1989). 
Effects of Endocrine Disrupting Chemicals in Wildlife 

Recently, a great deal ot of interest has arisen from the potential effects of 
endocrine disrupting chemicals (EDCs) on wildlife and humans. In fact, endocrine- 
disrupting effects of environmental contaminants have been observed or suspected in 
almost all taxa, ranging from invertebrates (gastropods) to fish, reptiles, birds, and 
mammals. At least 45 chemicals or their metabolites have been suggested as having 
endocrine-modulating activity that could lead to adverse population-level effects in 
wildlife (Colborn et al. 1993). Some of these chemicals include PCBs, DDT, dioxins, 
furans, and heavy metals. In addition, there is recent evidence indicating that exposure of 
fish to complex mixtures such as effluents discharged by sewage and paper mills, can also 
lead to endocrine alterations (Matthiessen and Sumpter 1998). Although the exact 
mechanism for the endocrine alterations is largely unknown, most of the concerns have 
been towards the potential effects of estrogenic substances. These compounds are 
capable of mimicking the action of steroid hormones such as estradiol, thus acting as 
partial (weak) or complete estrogen agonists or antagonists (Matthiessen and Sumpter 
1998). It is important to recognize, however, that estrogen mimicking is only one of the 
many possible mechanisms of endocrine modulation. Besides affecting hormone action, 
EDCs can also cause endocrine alterations through changes in biosynthesis, transport, and 
metabolism of hormones. 



The Pulp and Paper Industry 

If no specific reference is given in the following text, it is assumed the 
information was obtained from either Commission of the European Communities 1989, 
US EPA 1995, Thompson and Graham 1997, Erickson et al. 1998, or US EPA Office of 
Air Quality Planning and Standards 1998. 
Introduction 

It is estimated that each American consumes an average of approximately 300kg 
of paper products each year, making the U.S. the largest worldwide producer of paper. 
The approximately 555 pulp and paper mills in the U. S. manufacture wood pulp, primary 
paper products (e.g. printing and writing papers and sanitary tissue), and paper board 
products (e.g. container board and boxboard) mainly through the use of cellulose fibers 
from timber. Pulp facilities are comprised of mills that produce only pulp (market pulp 
facilities, 10% of the total), plants that manufacture paper from pulp produced elsewhere 
(non-integrated facilities, 54%), and mills that produce both pulp and paper on-site 
(integrated facilities, 36%). 
Pulp and Paper Manufacturing Process Sequence 

Presently, paper is made out of four basic sources of fiber: hardwood (such as oak, 
maple, birch), softwood (pine, spruce, hemlock), recycled paper, and nonwood fibers 
(cotton, hemp, flax). Making paper involves five basic steps: (1) fiber furnish 
preparation and handling, which involves debarking, slashing, and chipping wood logs 
and later screening chips and secondary fibers; (2) pulping, consisting of chemical, semi- 
chemical, or mechanical breakdown of pulp into fibers; (3) pulp processing, which 



removes impurities and cleans and thickens the pulp mixture; (4) bleaching pulp through 
the addition of water and of different bleaching agents on a specific sequence; and finally 
(5) stock preparation, which involves mixing, refining, and adding wet additives with 
the objective of increasing the strength, gloss, and texture of the final paper product. 
Because most of the pollutant releases associated with pulp and paper mills occur at the 
pulping and bleaching stages, a detailed description of only these processes will follow. 
Pulping techniques 

One of the challenges of using wood as a source of pulp is that in addition to the 
cellulose fibers, it contains other components (lignin, hemicellulose, and extractives such 
as resins, turpentine, tall oil, and soap) that need to be removed for the production of 
good-quality paper products. Although hemicellulose and extractives are generally easy 
to remove, the removal of lignin is difficult and requires the implementation of some type 
of pulping technique. The various methods of pulping can be classified as mechanical, 
chemical, or a combination of the two. 

The purpose of mechanical pulping is to physically tear the cellulose fibers from 
the wood. The oldest method of mechanical pulping is groundwood pulping, and consists 
of pressing blocks of wood against a rotating stone and later washing the fibers away 
from the stone with water. More modern mechanical pulping techniques include refiner 
mechanical pulping (RMP) and thermomechanical pulping (TMP). Because the pulp 
produced by mechanical pulping is of low strength and quality, it is mainly used for short- 
lived products like newspapers, catalogs and tissue. Mechanical pulping provides pulp 
yields of over 90% and accounts for approximately 7% of pulp production in the U.S. 



The objective of chemical pulping is to dissolve the lignin bonds holding the 
cellulose fibers together. This is achieved by cooking/digesting the wood chips in 
aqueous chemical solutions at elevated temperatures and pressures. The choice of 
chemicals used in this cooking process, as well as the length of chemical treatment, are 
important factors affecting the strength, appearance, and quality of the final paper 
product. In contrast to mechanical pulping, chemical pulping produces long, strong and 
stable fibers. The two major types of chemical pulping currently used in the U.S. are the 
kraft and the sulfite processes. Presently, the kraft or sulfate process is clearly the most 
popular method of chemical wood pulping. Its popularity stems from its ability to 
produce a high strength pulp with low costs because chemicals are readily recovered and 
reused. Lignin removal is high (up to 90%) which allows high levels of bleaching 
without pulp degradation due to delignification. This process is also very flexible and can 
be used with many types of raw materials. A downside of this technique is that it 
produces a very dark brown pulp, which requires the use of extensive chemical bleaching 
(see below). The kraft process uses a sodium-based alkaline pulping solution (liquor) that 
consists of sodium hydroxide and sodium sulfide in 10% solution. This "white liquor" is 
mixed with the wood chips in a digester, with the output products being wood fibers 
(pulp) and a liquid that contains the dissolved lignin solids in solution with the pulping 
chemicals ("black liquor"). The black liquor then undergoes a chemical recovery process 
to regenerate white liquor for the first pulping step. The kraft process has a high pulp 
yield (converts about 50% of input furnish into pulp) and produces a very strong pulp 
used for manufacturing bags, wrapping paper, container boards, and towels. 



8 

The sulfite process uses a solution of sulfur dioxide and calcium bisulfite to 
degrade the lignin bonds. It is usually restricted to softwood and non-resinous species of 
furnish, and produces pulps that have less color than kraft pulps making the bleaching 
process easier. This process is used for the manufacturing of products of average strength 
and extreme brightness (such as toilet and facial tissues, napkins, and photographic 
paper). In the U.S. chemical pulping accounts for about 60% of pulp production, and 
approximately 95% of this is produced using the kraft process. 

Semi-chemical pulping combines both chemical and mechanical treatment of 
fibers. It consists of chemically treating the wood (using caustic soda, sulfite, or sulfide) 
prior to mechanical defibrering. Yields and pulp quality can vary depending on the type 
and extent of chemical pretreatment. The neutral sulfite semichemical pulping (NSSC) is 
the most frequently used semichemical pulping method. Semichemical pulping allows 
for the production of fibers of intermediate length and strength good for the 
manufacturing of cardboard and paperboard. This technique accounts for 5% of pulp 
production in the U.S. 
Bleaching techniques 

Bleaching is defined as a chemical process designed to increase the brightness of 
the pulp. Bleached pulps create paper products that are whiter, brighter, and softer. 
Approximately 50% of the paper products manufactured in the U.S. are bleached in some 
fashion. A major factor determining the bleaching potential of a particular pulp is its 
amount of lignin. Pulps with high lignin content (mechanical pulping) are difficult to 
bleach, whereas chemical pulps can be bleached more efficiently due to their low lignin 






content. Since most of the bleaching is done on chemical pulps, the following description 
will be focused only on this type of bleaching. 

Chemical pulps are bleached in bleach plants where the pulp is processed in 
generally three to five stages of bleaching and washing. Bleaching stages generally 
alternate between acid and alkaline conditions. In the acid phase, chemicals react with 
lignin increasing the whiteness of the pulp, and later alkaline extraction dissolves 
lignin/acid reaction products. The product is washed at the end to remove both chemical 
solutions. Chemicals used in the bleaching process include hypochlorite (E), elemental 
chlorine (C), and chlorine dioxide (D). Because bleaching of pulps with chlorine and 
chlorine derivatives results in the production of chlorinated pollutants such as dioxins, a 
recent major trend in the industry has been the reduction in both the types and amounts of 
such chemicals used for pulp bleaching. In fact, many European mills have developed 
bleaching processes that are totally chlorine free (TCF) and that use chemicals such as 
ozone, oxygen, hydrogen peroxide, peracetic acid, and enzymes as bleaching agents. The 
use of chlorine dioxide has also steadily increased relative to elemental chlorine due to its 
reduction in the formation of chlorinated organics. Also, significant improvements have 
been made to improve delignification in order to minimize dioxin formation while 
reducing bleach chemical usage. Some of these delignification technologies include 
extended delignification during kraft pulping, solvent pulping, and oxygen 
delignification. 
Pollution Outputs 

The pulp and paper industry has historically been considered a major consumer of 
natural resources and a significant contributor of pollutant discharges to the environment. 






10 

The stages of pulping and bleaching are considered the major sources of pollutant outputs 
to air, water, and land, most of these being released by bleached kraft mills (effluents 
released by these mills are referred to as Bleached Kraft pulp Mill Effluents or BKME). 
The process of making pulp and paper is characterized by an intensive use of water. 
Indeed, the pulp and paper industry is the largest industrial water user in the U. S., with 
an average industry total discharge of 16 million m 3 /day of water. The main water 
pollution concerns are total suspended solids (TSS), biological oxygen demand (BOD), 
chemical oxygen demand (COD), total organic carbon (TOC), color, and turbidity. In 
addition, toxicity concerns arise from the presence of chlorinated organic compounds 
such as dioxins, furans, and others (collectively referred to as adsorbable organic halides 
or AOX) after the chlorination sequence. Recently, additional concerns have arisen from 
the potential toxic effects of natural components of wood (resin and fatty acids, and 
phytosterols) on aquatic organisms. 
Wastewater Treatment Technology 

It was estimated that during 1993, the pulp and paper industry produced about 2 
trillion pounds of waste. About 90% of this waste was managed on-site through recycling 
(5% of the total), energy recovery (10%), or treatment (75%). Pulp and paper mill plants 
in the U.S. operate treatment facilities (primary, secondary and/or tertiary) to remove 
BOD, TSS, and other pollutants (such as AOX) before discharging their effluents into a 
receiving waterway. Primary treatment mainly involves the mechanical removal of 
suspended solid fibers through sedimentation. Secondary treatment relates to biological 
degradation of effluents mainly through the use of aerated stabilization basins or 
oxygenated activated sludge. Both methods are based on accelerating nature's process of 



11 

reducing wastes to carbon dioxide and water using aerobic microorganisms, which will 
lead to significant reductions in BOD. Tertiary treatments use chemicals (such as ferric 
and aluminum oxide) to help increase the quality of the effluent being released. 
The Pulp and Paper Cluster Rules 

The pulp and paper cluster rules were promulgated in 1998 by the U.S. EPA as a 
way to simplify compliance by coordinating the regulation of industrial pollution. The 
major goals of this coordinated regulator approach are to provide a greater protection of 
human health and the environment; to reduce the costs of complying with wastewater and 
air emission regulations; and to promote and facilitate pollution prevention. These rules 
consist of regulations that specify both air emission standards (through the national 
emission standards for hazardous air pollutants (NESHAP)) and water effluent discharges 
(through the effluent limitations guidelines and standards, pretreatment standards, and 
new source performance standards). In general, the NESHAP requires mills to collect 
and control pulping and bleaching processes vent emissions and to eliminate the use of 
certain bleaching chemicals. Effluent regulations include best management practices to 
prevent leaks and spills of pulping liquor; specification of new analytical methods for 12 
chlorinated phenolics pollutants and for AOX; and a voluntary advanced technology 
program designed to encourage mills to install more pollution prevention technology than 
required by regulations. 









12 
Georgia-Pacific's Paper Mill Plant in Palatka, Florida 

General Description 

The Palatka paper mill plant has been in operation since 1947. This mill has two 
bleaching lines (40% product) and an unbleached line (60% product), which together 
release an estimated 36 million gallons of effluent daily. Treated effluents are discharged 
into Rice Creek, a small tributary of the St. Johns River. Rice Creek runs for about 5km 
prior to its confluence with the St. Johns River. Because Rice Creek is a small tributary, 
effluents can account for a large portion of its total flow (yearly average effluent 
concentration is estimated to be around 60%, with a range of 50% to 97%) (Myra 
Carpenter, personal communication). By the time effluents reach the St. Johns River, 
concentrations have fallen below 10%. It should be noted however, that these 
concentrations are higher when compared to the majority of paper mills in the U.S., 
where average effluent dilutions range from less than 1% to about 5%. 

In this plant, the bleaching sequences for the bleach lines are CEHD and 
CodioEopHDp, where Cd = mixture of chlorine (C) and chlorine dioxide (d) in 
proportions designated by subscripts; Eop = extraction with alkali and the addition of 
elemental oxygen (o) and hydrogen peroxide (p); H = hypochlorite; and Dp = 100% d 
substitution with the addition of p. The bleaching lines manufacture paper towels and 
tissue paper, whereas the unbleached line produces mainly kraft bag and linerboard. The 
wood furnish of this mill consists typically of 50% softwood species (mainly loblolly, 
slash, sand, and pine) and 50% hardwood (mainly tupelo, gums, magnolia, and water 
oaks). At the time of this study, effluents received secondary treatment, which consisted 






13 

of both anaerobic followed by aerobic biological degradation during a retention period of 

40 days. 

It should be noted that the bleaching sequence employed in the Palatka Operation 
does not represent about 75% of the mills in the U. S. This is because of basic toxicity 
concerns related to chlorinated phenolics and other chlorinated species, which prompted 
most mills to eliminate elemental chlorine bleaching and replace it with an elemental 
chlorine free (ECF) process using 100% chlorine dioxide in the first stage of bleaching. 
Data has shown that conversion to ECF can reduce water quality concerns substantially. 
Therefore, using the Palatka mill as a model of paper mill effluent effects should be 
considered a "worst-case" scenario. 
Ongoing Improvements 

Georgia-Pacific has studied what mill improvements will be necessary to comply 
with the U. S. EPA cluster rule promulgated in 1998. Some improvements that will be 
implemented in the next few years include the use of chlorine dioxide bleaching instead 
of elemental chlorine and also use of oxygen and hydrogen peroxide bleaching instead of 
sodium hypochlorite. In addition, improvements of secondary treatment of effluents to 
reduce BOD are currently underway. 

Sublethal Physiological Effects of Pub and Paper Mill Effluents on Fish 

Over the past 25 years, considerable effort has been devoted to determining the 
nature and extent of fish responses to pulp and paper mill effluents. The following is a 
brief review of sublethal effects (ranging from the biochemical to the organism level) 
measured in fish exposed to these complex effluents. For the purpose of this review, 






14 






responses have been grouped into the categories of general, liver, and reproductive health 

effects. 

General Health Effects 

Growth 

Growth can be considered as one of the ultimate indicators of health because it 
integrates most of the biotic and abiotic variables acting on an organism (Goede and 
Barton 1990). Laboratory experiments have shown that paper mill effluents can 
negatively affect growth rates in fish (Warren et al. 1974). Similarly, Munkittrick et al. 
(1991) reported that white suckers (Catostomus commersoni) collected from a site 
receiving primary-treated BKME were shorter, lighter, and grew slower than fish from 
reference sites. In contrast, Swanson et al. (1992) found no differences in growth rates 
between contaminated and reference populations of longnose sucker {Catostomus 
catostomus) and mountain whitefish (Prosopium williamsoni), and Servizi et al. (1992) 
reported no effects of treated BKME on growth of Chinook salmon (Oncorhynchus 
tshawytscha) in the laboratory. 
Hematology 

Hematology is defined as the study of blood and blood-forming or hematopoietic 
tissues (primarily spleen and kidney in fish). Fish exposed to paper mill effluents may 
respond by either decreasing (anemia) or increasing (polycythemia) several hematological 
variables. Many field and laboratory studies have reported anemia in fish due to a decline 
in the number of red blood cells and/or in hemoglobin concentrations after exposure to 
BKME (Everall et al. 1991, Swanson et al. 1992, Khan et al. 1996, Soimasuo et al. 









15 

1998). It has been postulated that declines in hemoglobin may result from increased 
breakdown of red blood cells (hemolysis), since this phenomena has been induced in vitro 
after exposure of red blood cells to resin acids (Bushnell et al. 1985). Increases in 
hematocrit values probably due to disturbances in ion regulation and/or to stress-induced 
polycythemias have also been reported in fish sampled downstream from paper mills 
(Oikari et al. 1985, Hodson et al. 1992) and in fish exposed to chlorinated compounds 
present in BKME (Bengtsson et al 1988). In a field study on the effects of BKME 
exposure on perch (Percafluviatilis), although there was a decline in hemoglobin 
concentrations in polluted stations, this decline was associated with an increase in the 
number of red blood cells (Larsson et al. 1988). These authors concluded that this 
increased erythropoiesis was likely due to an increased oxygen demand as a response to 
the high detoxification activity associated with exposure to these effluents. Several 
studies, however, have found no effects on hematology of fish exposed to BKME. For 
instance, chronic exposure (210 days) of Chinook salmon to treated BKME (0.3 to 4% 
v/v) had no effect on hematocrit (Servizi et al. 1992), and Kennedy et al. (1995) found no 
changes in hemoglobin in rainbow trout (Salmo gairdneri) exposed to resin acids for 24 
hours. Finally, Swanson et al. (1992) reported no differences in packed cell volume 
between fish sampled close to a paper mill area and reference fish. 
Spleen histology 

The spleen is one of the primary hematopoietic organs in fish, and thus 
histological alterations in this tissue could explain some of the hematological changes 
described above. Hemosiderosis (accumulation of hemosiderin, an endogenous pigment 






16 

that results after the breakdown of hemoglobin, within the spleen cells) has been observed 
in spleens of fish naturally exposed to BKME (Khan et al. 1992, Mercer et al. 1997). 
Osmoregulation 

Structural changes in the gills have been observed in fish exposed to BKME, 
which may lead to disruptions in oxygen diffusion and osmoregulatory functions. The 
literature on this subject is confusing, however, since a wide array of electrolyte changes 
(increases, decreases, and no effects) have been reported in fish exposed to BKME 
(Oikariefa/. 1988, Larsson et al. 1988, Lindstrom-Seppa and Oikari 1989, 1990, 
Lehtinen et al. 1990, Everall et al. 1991, Swanson et al. 1992, Jeney et al. 1996). 
Immune function 

Exposure of fish to BKME may increase the circulating levels of corticosteroids 
leading to immunological system disruptions, such as reductions in leuccorit and in 
immunoglobulins (Jokinen et al. 1995, Soimasuo et al. 1995a, 1995b, Khan et al. 1996). 
These changes in turn can result in an increased susceptibility to pathogens such as 
bacteria and parasites. Kennedy et al. (1995) exposed juvenile trout to sublethal 
concentrations of chlorinated resin acids for 24 hours and observed a reduced resistance 
to infection by Aeromonas salmonicida. Several studies have also reported an increase in 
the prevalence and intensity of infection with ecto and endoparasites in fish exposed to 
pulp and paper effluents (Thulin et al. 1988, Axelsson and Norrgren 1991, Khan et al. 
1992, 1994b). 



17 

Liver Health Effects 

Because the liver is the primary organ for the biotransformation and excretion of 
xenobiotics, the evaluation of its health and functioning is fundamental in any study on 
the effects of environmental contaminants. Some of these measurements include analyses 
of liver enzyme activities and evaluations of liver weights and histology. Biochemical 
responses of fish to chemical stimuli have been studied extensively over the past years, 
and the increase in monooxygenase enzyme activity (measured as ethoxyresorufin-O- 
deethylase or EROD activities) in fish livers sampled downstream of BKME is a good 
example of this. One of the major advantages of using biochemical responses as 
indicators of contaminant exposure is their high sensitivity and rapid response time. A 
major problem with this approach, however, is that the exact biological significance of 
these changes for the functional integrity of the organism is poorly known (Thomas 
1990). In addition, factors such as temperature, age, sex, and nutritional status of fish can 
modify the activity of these detoxification enzymes, which could complicate the 
interpretation of induction responses in fish (Jimenez and Stegeman 1990). 
Liver enzymes 

Cytochrome P450 refers to a family of enzymes involved in the biotransformation 
of organic chemicals, resulting in either their activation to toxic metabolites or their 
inactivation. P450 systems in fish are inducible by different types of endogenous and 
exogenous compounds, a process that involves synthesis of new messenger RNA, and 
thus of new enzyme protein (Stegeman et al. 1992). Since EROD activity is catalyzed by 
P450 monooxygenases, an increase in EROD activity is indicative of P450 induction. 
Measurements of EROD activity have been widely used as a biomarker for exposure of 



18 

fish to several groups of chemicals, including polychlorinated dibenzo-p-dioxins 
(PCDDs) and dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polycyclic 
aromatic hydrocarbons (PAHs), pesticides, metals, and natural biogenic substances. 
Because BKME have been reported to contain EROD-inducing compounds, this 
biomarker has played a major role in the study of fate and biological effects of paper mill 
effluents. In general, researchers have reported background EROD activities in fish from 
reference sites, with significant increases in areas close to pulp mill outfalls (Forlin et al. 
1985, Lindstrom-Seppa and Oikari 1989, Courtenay et al. 1993, Bankey et al. 1994, 
Soimasuo et al. 1995b). Until recently, it was believed that the main inducers in mill 
effluents were chlorinated persistent compounds (such as PCDDs and PCDFs) (Hodson 
1996). However, new evidence suggests that enzymatic EROD induction also occurs in 
fish exposed to unbleached effluents, and that the compound(s) responsible for such 
induction are not of the highly hydrophobic chlorinated type, but rather of the moderately 
hydrophobic planar PAH-type form present as natural components of wood, and readily 
metabolized by fish (Hodson 1996). 

The effect of treated vs. untreated paper mill effluents on EROD activity in fish 
has only recently been addressed and the results are so far inconclusive. Martel et al. 
(1996) found that 17 of 46 primary and secondary-treated paper mill effluents did not 
cause significant mixed-function oxygenase (MFO) responses in fish. In a later study, 
Martel and Kovacs (1997) reported a significant increase in EROD activity in rainbow 
trout exposed to primary-treated effluent compared to fish exposed to secondary-treated 
effluents. In contrast, a field study revealed that EROD induction in wild fish was not 
eliminated after the installation of a secondary treatment facility (Munkittrick et al. 



.9 

1992a). Also, wild European carp (Cyprinus carpio) exposed to treated pulp mill 
effluents had higher elevated hepatic EROD levels relative to reference fish (Ahokas et 
al. 1994). In the latter study, EROD activity was strongly correlated with water AOX 
levels, and poorly related with fish muscle and sediment extractable organic halogen 
(EOX) levels. Gagne and Blaise (1993) also noted that EROD activity in rainbow trout 
increased in fish exposed to sublethal concentrations of both primary and secondary- 
treated effluents, but that the degree of increase was higher in the primary-treated exposed 
group. 

Phase II (conjugating) enzymes have also been studied in fish exposed to BKME. 
The two most important conjugating enzymes studied include glutathione S-transf erases 
(GSTs) and UDP-glucoronosyltransferases (UDPGT). Cytoplasmic GSTs are a multi- 
gene family of proteins that participate in detoxification processes by conjugating many 
electrophilic compounds with glutathione (GSH) to produce more soluble and thus 
excretable products (George and Buchanan 1989). Studies on the detoxification capacity 
of effluent-exposed fish have reported both increases (Oikari et al. 1988) and declines 
(Mather-Mihaich and Di Giulio 1991, Bucher et al. 1993) in hepatic GSH concentrations. 
GST activity, on the other hand, has generally been found unaltered after exposure to 
BKME (Soimasuo et al. 1995a, 1995b). 

UDP-glucoronosyltransf erase enzymes catalyze the transfer of glucoronyl groups 
from uridine 5'-diphosphoglucuronate to many acceptors including PAHs and various 
endogenous compounds (Stegeman et al. 1992). Field and laboratory studies on the 
effects of BKME on fish have reported inductions, decreases, and no effects on UDPGT 
activity (Forlin et al. 1985, Lindstrom-Seppa and Oikari 1988, Lindstrom-Seppa et al. 



20 

1989). Inhibitory effects on UDPGT activity (by up to 85%) have been observed in 
rainbow trout after exposure to trichlorophenol, pentachlorophenol, and dehydroabietic 
acid (all common components of paper mill effluents) (Andersson et al. 1988). This type 
of inhibition can have important consequences, since it can not only reduce the ability of 
UDPGT to metabolize xenobiotics but may also affect its role in metabolizing 
endogenous compounds. This could explain the increase in bilirubin (a substrate of 
UDPGT) in blood of BKME-exposed fish (Oikari and Nakari 1982). 
Carbohydrate metabolism 

Disturbances in carbohydrate metabolism have been observed in fish exposed to 
BKME. It has been postulated that these effluents are capable of causing internal hypoxia 
thorough gill damage (Davis 1973), which can lead to increased blood glucose levels and 
depletion of liver glycogen. Exposure of coho salmon (Oncorhynchus kisutch) to an 
effluent concentration equivalent to 0.8 of the 96-h LC50 produced an immediate 
hyperglycemia, and after 48h of exposure liver glycogen concentrations had decreased to 
almost zero (McLeay and Brown 1975). In another study, Oikari and Nakari (1982) 
exposed trout to components of paper mill effluent for 1 1 days and observed an 
exhaustion of liver glycogen reserves. 

Blood glucose is one of the most commonly used parameters utilized for 
measuring stress. The classic stress response involves an elevation of blood sugar in 
response to the hormones adrenaline and Cortisol. In rainbow trout exposed to 
chlorinated phenolics and resin acids, plasma glucose concentrations were higher 
compared to control fish, and concentrations remained high throughout the 40-day 
experiment (Tana 1988). Hyperglycemia has also been reported from perch sampled 



21 

from an area contaminated with paper mill effluents (Andersson et al. 1988) and from 
rainbow trout artificially exposed to chlorinated resin acids (Kennedy et al. 1995). Some 
studies, however, have failed to detect changes in liver glycogen and/or blood glucose 
concentrations in fish after exposure to BKME (Oikari et al. 1988, Swanson et al. 1992, 
Soimasuo et al. 1998). 
Liver histology 

Livers of male bullheads (Cottus gobio) sampled close to an area affected by 
paper mill effluents had a high incidence of fatty degeneration, fibrosis, necrosis, and 
parasitism (Bucher et al. 1992). Khan et al.{ 1994a) also found that winter flounder 
(Pleuronectes americanus) taken from areas contaminated with BKME had livers with 
varying degrees of vacuolation and multifocal hemosiderosis. Similarly, in three-spined 
stickleback (Gasterosteus aculeatus) chronically exposed to pulp mill effluents (5 1/2 
months) several anomalies in the liver were observed (necrosis, nuclear pyknosis, 
vacuolation, and fat accumulation) (Axelsson and Norrgren 1991). Servizi et al. (1992) 
reported an increase in the incidence of hepatic granulomas in Chinook salmon 
chronically exposed (for up to 210 days) to treated BKME. In contrast, Mather-Mihaich 
and Di Giulio (1991) found no histopathological changes in liver of channel catfish 
(Ictalurus punctatus) exposed to BKME for up to 14 days, and Mercer et al. (1997) 
reported that 84% of cunner (Tautogolabrus adspersus) sampled at a reference site 
showed evidence of vacuolation in the liver in contrast to 53% from the vicinity of the 
paper mill. 



22 

Hepatosomatic index 

Ratios of organ weight to body weight have been reported by several authors 
when studying the effects of paper mill effluents on fish. The hepatosomatic index (HSI) 
is calculated by dividing the weight of the liver by the body weight of the fish and 
multiplying the resulting number by 100. Munkittrick et al. (1992a) reported that, after 
the initiation of secondary treatment of BKME, liver weights of lake whitefish 
(Coregonus clupeaformis) decreased by 37% in females and by over 50% in males 
compared to values found at reference sites. Somatic indices were also decreased in 
winter flounder inhabiting an inlet under the influence of pulp and paper mill effluent 
(Khan et al. 1992). Similarly, Bucher et al. (1992) reported a decrease ("shrinkage") of 
livers from bullheads sampled at the end of the low-water period in a river contaminated 
with BKME. In this study, enlarged livers were found only following the high-water 
period. The authors concluded that this change in liver weights was directly related to a 
change in glycogen content in the hepatocytes (Bucher et al. 1992). On the other hand, 
enlarged livers have also been reported in fish contaminated with paper mill effluents 
(Larsson et al. 1988, Andersson et al. 1988). These authors attributed the increase in HSI 
in BKME-exposed fish to proliferation of the endoplasmic reticulum, as well as to 
increased fat accumulation. 
Reproductive Health Effects 

From recent studies in several fish species, there is substantial evidence that 
exposure to paper mill effluents can result in reproductive alterations, such as delayed 
sexual maturation, altered secondary sex characteristics, reduced gonad weights, decline 
in the production of eggs and in their sizes, and decreased concentrations of sex steroids. 



23 

Although the compounds responsible for these effects have not yet been identified, it has 
been hypothesized that these changes might be related to exposure to natural components 
of wood (such as resin acids, sterols, and lignins), which have been reported to have weak 
estrogenic activity (Van Der Kraak et al. 1998). 

Results from studies on white sucker from Jackfish Bay, Canada, indicate that 
several sites within the pituitary-gonadal-axis are affected after exposure to BKME. Fish 
from exposed sites had significantly lower plasma levels of gonadotropin (GtH-II) and 
showed depressed responsiveness of sex steroids and 17,20B-dihydroxy-4-pregnen-3-one 
(17,206-P, a maturation-inducing steroid) after injections with gonadotropin releasing 
hormone (GnRH) (Van Der Kraak et al. 1992). BKME-exposed fish also had lower 
circulating levels of testosterone glucoronide, which would be suggestive of altered 
peripheral steroid metabolism. Similarly to what was observed under in vivo conditions, 
in vitro incubations of ovarian follicles collected from BKME-exposed females have also 
shown reduced production of testosterone, 17p-estradiol, and 17,206-P 2 under basal and 
human chorionic gonadotropin stimulated conditions (Van Der Kraak et al. 1992, 
McMaster et al. 1995). The similarities between both types of studies would suggest that 
reductions in plasma steroid levels in BKME-exposed fish from Jackfish Bay are mainly 
due to alterations in ovarian steroid production. 
Age at maturity 

This parameter is defined as the age (in years) in which spawning first occurs. In 
perch exposed to BKME, less than 50% of the potentially mature males had developed 
gonads, compared to 80% at the reference site (Sandstrbm et al. 1988). Similarly, lake 
whitefish and white sucker exposed to primary treated BKME exhibited delayed sexual 



24 

maturity relative to reference populations (Munkittrick et al. 1991, 1992a). In a study on 

the effects of paper mill effluents on longnose sucker and mountain whitefish, Swanson et 

al. (1992) found no differences in age at maturity between contaminated and reference 

populations. 

Secondary sex characteristics 

These are traits that distinguish males from females but that are not responsible 
for the production of gametes. Mature male white suckers naturally exposed to paper mill 
effluents showed no evidence of secondary sexual characteristics (nuptial tubercles) in 
relation to males sampled from a reference site (Munkittrick et al. 1991). Female 
mosquitofish, Gambusia affinis, inhabiting a stream receiving paper mill effluents in 
Florida were reported to be strongly masculinized showing both physical secondary sex 
characteristics (fully developed gonopodium) and reproductive behavior of males 
(Howell et al. 1980). More recently, masculinization of female fish has been identified 
from an additional two species (least killifish, Heterandria formosa and sailfin molly, 
Poecilia latipinna) collected from Rice Creek, the stream receiving the effluents 
discharged by the Palatka mill (Bortone and Cody 1999). Masculinization of female fish 
has been attributed to the action of androgenic hormones that result from the 
biotransformation of plant sterols (and also cholesterol and stigmasterol) by bacteria such 
as Mycobacterium (Howell and Denton 1989). 
Gonadosomatic index 

Changes in gonad weights in relation to body weights (gonadosomatic indices or 
GSIs) are routinely used as a way to assess reproductive effects in fish exposed to paper 
mill effluents and other environmental contaminants. Several studies have reported 



25 

declines in GSIs in fish exposed to BKME (Larsson et al. 1988, Munkittrick et al. 1991, 

1992a, 1994, Gagnon et al. 1994b, Gibbons et al. 1998). However, there is also evidence 

to suggest that decreases in gonadal size in response to declines in sex steroids may not 

always occur after exposures of fish to BKME (McMaster et al. 1996b), which would 

indicate differences in reproductive responsiveness to contaminant exposure across 

species. 

Fecundity and egg size 

There are relatively few studies on the effects of BKME on egg parameters, and 
the results from these studies are conflicting. Many field and laboratory studies have 
reported declines in fecundities of several fish species after exposure to paper mill 
effluents (Landner et al. 1985, Munkittrick et al. 1991, Gagnon et al. 1994b, 1995, 
Kovacs et al. 1995). Fecundities, however, were not altered after exposures to BKME in 
several other field (Karas et al. 1991, Swanson et al. 1992, Adams et al. 1992) and 
laboratory studies (Kovacs et al. 1996). Sandstrbm et al. (1988) reported that developing 
eggs from female perch sampled close to a bleachery outlet were smaller and more 
irregular in shape compared to controls. In lake whitefish naturally exposed to BKME, 
Munkittrick et al. (1992a) reported that even though females had a higher fecundity 
compared to females from a reference site, these eggs were smaller. McMaster et al. 
(1991) also reported reduced egg size in white sucker females exposed to BKME. 
Sex steroids 

The most important reproductive hormones in teleost fish are testosterone, 11- 
ketotestorene, and 176-estradiol. They are produced by the gonads and their 
measurement in plasma is a good indicator of reproductive status, seasonality, and 



26 

gonadal function. One of the most consistent findings in studies that have focused on the 
effects of BKME on reproductive parameters of fish is a decline in the concentration of 
sex steroids in plasma of exposed animals. BKME-exposed white suckers from Jackfish 
Bay, Lake Superior show decreased concentrations of several sex steroid hormones 
(testosterone, 11-ketotestosterone 17B-estradiol, and 17, 20P-dihydroxy-4-pregnen-3-one) 
(Munkittrick et al. 1991, McMaster et al. 1995, 1996b). Declines in steroid 
concentrations have also been documented in longnose sucker and lake whitefish from 
Jackfish Bay (Munkittrick et al. 1992a, McMaster et al. 1996b), in white sucker at other 
mills (Hodson et al. 1992, Munkittrick et al. 1994, Gagnon et al. 1994a), and in other fish 
species sampled elsewhere (Adams et al. 1992, McMaster et al. 1996b). The 
consequences of these similar endocrine alterations to whole animal reproductive fitness 
and population dynamics, however, have varied greatly between species. For example, 
longnose sucker exposed to BKME show no organism responses other than an altered age 
distribution, whereas white sucker and lake whitefish show decreased gonadal sizes, 
secondary sexual characteristics, and egg sizes, and increased age to maturity (McMaster 
et al. 1996b). In a review of whole organism responses of fish exposed to different kinds 
of mill effluents (including unbleached pulps), 48% of the populations studied had 
increased condition factors, 80% showed increased age to sexual maturation, and reduced 
gonadal size was reported in 58% of the studies (Sandstrom 1996). These observations 
provide evidence for species differences in susceptibility to BKME, but also show the 
inherent difficulty when trying to compare biological responses in fish populations 
inhabiting highly different environments and exposed to complex mixtures likely to vary 
in chemical composition. 



27 

Vitellogenin 

Vitellogenesis involves the synthesis of vitellogenin by the liver, its uptake by 
growing oocytes, and its storage as yolk to serve as source of food for the developing 
embryos. There is little information on the effects of BKME on plasma vitellogenin 
concentrations. Plant sterols, such as p-sitosterol, which are commonly found in pulp 
mill effluents, are estrogenic compounds known to bind in vitro to rainbow trout hepatic 
estrogen receptors (Tremblay and Van Der Kraak 1998) and can induce vitellogenin 
synthesis in male goldfish (Carassius carassius) (MacLatchy and Van der Kraak 1995). 

The Largemouth Bass (Micropterus salmoides) 

This section outlines some important biological information regarding the fish 
species used as the study model during the course of this dissertation. Except for the 
information presented under general description and geographical distribution, this 
summary is focused on presenting data related to the Florida subspecies of largemouth 
bass. 
General Description 

There are two recognized subspecies of largemouth bass: the Florida (M. 
salmoides floridanus) and the northern (M. salmoides salmoides) subspecies. 
Morphologically, they are both very similar with the major differences being a larger 
number of scale rows and pyloric caeca in the Florida subspecies (Bailey and Hubbs 
1949). Largemouth bass are characterized by having a robust and compressed body that 
can measure up to 876 mm in length, and a long head and mouth that contains brush-like 
teeth on both jaws and that extend to cover the palatines, vomer and pharynx (Hardy 



28 

1978). Their pigmentation is dark brown to olive with a silvery sheen and a conspicuous 
black lateral stripe that fades with age (Chew 1974). 
Geographic Distribution 

In North America, the northern largemouth bass has a wide geographic 
distribution ranging from Mexico to southern Canada (Hardy 1978). In the U. S., they 
can be found in many states including Virginia, West Virginia, Texas, Oklahoma, Kansas, 
Nebraska, Iowa, Minnesota, Wisconsin, North Dakota, New York, Pennsylvania, Ohio, 
Maryland, and Florida (Hardy 1978). Although M. salmoides floridanus is endemic to 
Florida, in northern areas of the state its range overlaps with that of M. salmoides 
salmoides (Bailey and Hubbs 1949). 
Habitat and Range 

In Florida, largemouth bass can be found in most habitats (lakes, ponds, bayous, 
marshes, sloughs, impoundments, rivers, and creeks), except some low-oxygen boils. 
They occur over all types of substrates (mud, muck, organic debris, sand, clay, and 
gravel) and depths, but prefer shallow, vegetated areas (water lilies, cattails, and pond 
weed) (Chew 1974, Hardy 1978). Known water quality parameter ranges under field 
conditions for the species are: 0.56 - 35.0°C for temperature (with an optimum of 26.6 - 
27.7°C); 4.7 - 1 1.0 for pH; and a maximum salinity of 32.1 ppt (Hardy 1978). 

Information on the range of movement of Florida largemouth bass suggests the 
presence of both mobile and sedentary populations. Snyder et al. (1986) reported that 
38% of the bass marked and released in the lower St. Johns River were recaptured in the 
same area are as tagged, and of the remaining 62%, 44% had moved a distance of less 






29 

than 2km. In another study, 84% of specimens tagged moved less than 8km, with a 
maximum distance of 20km (cited by Hardy 1978). 
Growth and Feeding Habits 

Growth rates in Florida largemouth bass are higher when compared to their 
northern counterpart, probably due to both intrinsic factors as well as to the more 
favorable environment and extended growing season present in southern latitudes 
(Clugston 1964). In this respect, fry growth is directly related to water temperature, with 
minimum and maximum growth rates at temperatures below 17.5 and above 25°C, 
respectively. In addition, growth rates are known to vary with age (are highest during the 
first two years and decreases after fish reach sexual maturity) and season (are highest in 
summer and fall and lowest in winter and spring) (Chew 1974). For example, in bass 
from Lake Weir, Florida, growth rates from hatching to 1 year of age, and from ages 1 to 
2, were estimated at 0.383mm/day and 0.255mm/day, respectively (Chew 1974). 

There is a well documented shift in the diet of largemouth bass in relation to age. 
Approximately 40 hours after they leave the nest, larvae feed almost entirely on 
crustaceans (mainly copepods and cladocerans) (Kramer and Smith 1960). By the time 
bass reach 50mm, insects and fish are the next prey items to become incorporated in the 
diet. Studying the food habits of largemouth bass inhabiting the St. Johns River, Mclane 
(1949) also demonstrated a progressive change in diet, from zooplankton and macro- 
invertebrates (cladocerans, decapods, and insects (larvae, pupae, and nymphs)) in fry and 
juveniles, to almost exclusively fishes in adults. 



30 









Reproduction 
Sexual maturity 

In Florida, largemouth bass reach sexual maturity at a size of 250mm, i.e. within 
the first year of age. Minimum size at maturity has been reported at 140mm. A restricted 
growing season in northern climates precludes attainment of sexual maturity in one year, 
and extends it to 2-4 years of age instead (Hardy 1978). 
Spawning 

Florida largemouth bass are capable of successfully spawning anywhere from 
mid-November through August, with peaks in February and March (Clugston 1966). 
Induction of spawning is mainly triggered by increases in water temperature during the 
spring. Spawning usually occurs near dusk or dawn, with maximum activity at water 
temperatures between 20 and 24°C and no spawning has been observed at temperatures 
below 18°C and above 27°C (Clugston 1966). 

Nests are built in 0.6 to 1.2m of water, although depths can range from 10cm to 
over 2m (Carr 1942). Males of the species are in charge of constructing the nests usually 
through excavation of substrate, although in some instances no nest is prepared and eggs 
are deposited directly on aquatic vegetation. Nests are often built in open areas in 
association with various aquatic plants and on different types of substrates ranging from 
fibrous organic debris to bare sand (Kramer and Smith 1960). Nests measure from 30.5 
to 152.4cm, are round, and are located between 1.2 and 6.4m offshore at spacing intervals 
of 1.8 to 2.1m (Carr 1942, Hardy 1978). Careful spacing of nests is related to the strong 
territorial behavior exhibited by males during the spawning season. Fecundity is highly 



31 

variable ranging from 2,000 to 145,000 eggs, and appears to be directly related to age and 
condition of the fish, as well as to environmental parameters such as water temperature 
(Chew 1974, Hardy 1978). 
Reproductive cycles 

As the spawning season approaches, the percent of gonad weight to total body 
weight (the gonadosomatic index or GSI) increases. In Florida largemouth bass (age 
class I) reared in Texas, GSIs peaked in March in females (to about 5%), and at a slighter 
later date (late April) in males (to about 0.9%) (Rosenblum et al. 1994). In northern 
largemouth bass from Tennessee the peak in female GSIs occurred later, between mid 
April (age class n, to about 5%) and mid May (age class I, to about 5.5%) (Adams and 
McLean 1985). Seasonal changes in organ somatic indices of M. salmoides floridanus 
(GSI and hepatosomatic index or HSI) have also been observed in bass sampled from 
lakes in central Florida, with highest values in January and February (Timothy Gross, 
unpublished data). Seasonal cycles of sex steroids and vitellogenin have also been 
studied in females from Lake Woodruff, Florida (Timothy Gross, unpublished data). This 
study reports peaks in 17P-estradiol, testosterone, and 1 1-ketotestosterone in February at 
concentrations of 3,892, 2,167, and 971 pg/mL, respectively. Vitellogenesis, on the other 
hand, begins in September and peaks in January with values of 6.3mg/mL of vitellogenin 
in plasma (Timothy Gross and Nancy Denslow, unpublished data). As with other teleost 
species, the predominant sex steroids in female and male largemouth bass during the 
reproductive season are 1 7p-estradiol and 11-ketotstosterone, respectively, and 
vitellogenin in females is usually found at concentrations that are about 12 times the 
values reported in males. 



32 

Eggs and fry 

The eggs of the largemouth bass are spherical or oval, adhesive and demersal. 
They are light yellow to orange, and contain one large oil globule that measures 0.34 - 
0.54mm in diameter and that persists throughout the entire embryonic and larval stages 
until the yolk is completely reabsorbed (Chew 1974, Hardy 1978). Unfertilized eggs 
measure between 0.75 - 1.7mm in diameter, whereas fertilized eggs are larger measuring 
between 1.3 and 1.95mm (Hardy 1978). After deposition, eggs always lie with the oil 
globule uppermost, and water-harden within 15 minutes (Carr 1942). Embryonic 
development can be summarized as follows: first mitotic divisions of the ova begin about 
one hour after fertilization; blastula stage is reached at 3.25hr; blastoderm at 5.25hr; 
gastrula at 14hr; early embryo at 21.5hr; and late embryo stage occurs at 37hr post- 
fertilization (Chew 1974). Under laboratory conditions, hatching has been reported to 
begin between 45 and 47hr after fertilization (Carr 1942, Chew 1974). Larvae are highly 
active about 77hrs post-fertilization, and can swim off the bottom about 73hrs later. The 
mouth is fully formed after 167hrs, and initial feeding has been observed after 206hrs, or 
approximately on the 8 th day, although the yolk is not yet fully absorbed at this time 
(Chew 1974). This sequence of events agrees with what has been reported to occur under 
field conditions. For example, Carr (1942) found that in Lake Bivans Arm, Florida, eggs 
hatched 50 to 60hrs after fertilization, and larvae began to leave the bottom 4 days after 
hatching. In this study, regular feeding and schooling was recorded on the 8 l day. In 
another study, rise from the nest was reported to occur at fry lengths of 5.92 - 6.31mm 
(average of 6.16mm) (Kramer and Smith 1960). Time of rising from the nest is inversely 



33 

correlated with temperature (from 7.2 days at temperatures between 13 and 18°C, to 6.0 

days at temperatures ranging from 21 to 24°C) (Hardy 1978). 

Survival 

Kramer and Smith (1962) found that the success of a year-class of largemouth 
bass was determined within the first two weeks of spawning. They concluded that the 
major mortality factors at this time were related to water temperature and wind (major 
drops in water temperature and strong winds caused high mortalities). They also found 
that food availability, predation, and fecundity of the spawning stock did not play a major 
role in the observed mortalities. 

Significance of this Work 

Field experiments provide data that is easily related with the natural occurring 
studied phenomena (external validity) while laboratory experiments provide the settings 
to adequately control the effects of confounding variables (internal validity). Because of 
this trade-off between external and internal validity the effects of BKME on health and 
reproduction of largemouth bass were studied through paired laboratory and field studies. 

The primary objective of the field studies was to determine whether reproductive 
and health parameters were altered in fish populations inhabiting streams contaminated 
with BKME. The primary objective of the experimental studies was to determine dose- 
related effects in captive fish exposed to paper mill effluents. For these experiments, 
effluent concentrations were calculated based upon the current range of environmental 
concentrations of effluent reported for Rice Creek and the St. Johns River (10 - 90%). 
Exposure periods were designed to reflect multiple endpoints throughout the reproductive 



1A 






season. Exposure of BKME at concentrations likely to be encountered by free-ranging 
fish over a given period is fundamental for any risk assessment. In addition, most toxic 
effects are based on lethality. However, sublethal effects, such as effects on growth and 
development, liver function, and other physiological parameters are equally if not more 
important when trying to determine the effects of BKME on fish. In addition, an 
understanding of the sublethal effects of BKME on the reproductive physiology of adult 
fish is essential for evaluating the impact of these and other environmental contaminants 
at a population level. 

Organization of Dissertation 

This dissertation evaluates the effects of BKME on health and reproduction of 
largemouth bass. Specifically, the objectives of this work are as follows: 

• To compare reproductive parameters of Florida largemouth bass sampled from 
reference and BKME-exposed sites along the St. Johns River (Chapter 2). 

• To expose adult largemouth bass to different concentrations of BKME for various 
lengths of time and evaluate effects on reproductive physiology (Chapter 3). 

• To evaluate the effects of BKME exposure on health parameters of largemouth bass 
through the conduction of both field and laboratory studies (Chapter 4) 

• To expose adult largemouth bass to different concentrations of BKME for various 
lengths of time and evaluate effects on reproductive physiology and success (Chapter 

5). 

• To examine the effects of BKME and resin acids on the steroidogenic capacity of 
isolated ovarian follicles (Chapter 6). 



35 

• To integrate these findings, evaluate their ecological significance, and propose areas 
of future research needs (Chapter 7). 



CHAPTER 2 

COMPARISON OF REPRODUCTIVE PARAMETERS FROM FLORIDA 

LARGEMOUTH BASS (MICROPTERUS SALMOIDES FLORIDANUS) SAMPLED 

FROM REFERENCE AND CONTAMINATED SITES FN THE ST. JOHNS RIVER 

AND TRIBUTARIES 



Introduction 

Over the past decade, several Canadian and Scandinavian studies have focused on 
the effects of bleached kraft pulp mill effluent (BKME) on multiple biochemical and 
physiological parameters of fish. From these studies, some of the most meaningful 
responses have been related to altered reproductive function. Specifically, fish exposed 
to BKME have lower circulating concentrations of reproductive hormones (testosterone, 
11-ketotestosterone, and 17B-estradiol), reduced gonadal growth, increased age to sexual 
maturation, smaller eggs, and reduced expression of secondary sex characteristics when 
compared to fish from reference sites (Sandstrom et al. 1988, Larsson et al. 1988, 
Andersson et al. 1988, Munkittrick et al. 1991, 1992b, 1994, McMaster et al. 1991). 

Detailed endocrine laboratory studies have demonstrated that the pituitary-gonadal 
axis of fish is affected by exposure to BKME, including decreased circulating 
concentrations of gonadotropin (GtH) and sex steroids, depressed responsiveness of 
gonadal steroidogenesis to gonadotropin-releasing hormone (GnRH), and altered 
peripheral metabolism of sex steroids (Van Der Kraak et al. 1992). In addition, 
McMaster et al. (1995) reported a reduced conversion of testosterone to 178-estradiol, 
indicating a reduced level of aromatase in BKME-exposed follicles during early 

36 






37 

vitellogenic stages. In another study, steroid synthesis by ovarian follicles from BKME- 
exposed and non-exposed female white suckers (Catostomus commersoni) was similar, 
suggesting that the origin of different steroid concentrations in wild BKME-exposed fish 
is external to the gonad (Gagnon et al. 1994b). 

Effects of paper mill effluents on the reproductive physiology of fish have also 
been documented in the St. Johns River, Florida. Female mosquitofish, Gambusia 
affinis, inhabiting a stream receiving paper mill effluents were strongly masculinized 
showing both physical secondary sexual characteristics (fully developed gonopodium) 
and reproductive behavior of males (Howell et al. 1980). Masculinization of female 
mosquitofish has also been reported from laboratory studies after exposures to 6- 
sitosterol, a plant sterol byproduct of wood delignification (Denton and Howell 1989). 
More recently, masculinization of female fish has been identified from an additional two 
species (least killifish, Heterandria formosa and sailfin molly, Poecilia latipinna) 
collected from paper mill effluent-receiving streams (Bortone and Cody 1999). 

Since 1947, the St. Johns River has received effluents from a paper mill plant 
located in Palatka (Figure 2.1). This mill has two bleaching lines (40% product) and an 
unbleached line (60% product), which together release an estimated 36 million gallons of 
effluent daily. In this plant, the bleaching sequences for the bleach lines are CEHD and 
CgodioEopHDp (where Cd = mixture of chlorine (C) and chlorine dioxide (d) in 
proportions designated by subscripts; Eop = extraction with alkali and the addition of 
elemental oxygen (o) and hydrogen peroxide (p); H = hypochlorite; and Dp = 100% d 
substitution with the addition of p). The bleaching lines manufacture paper towels and 
tissue paper, whereas the unbleached line produces mainly kraft bag and linerboard. The 



38 

wood furnish of this mill consists typically of 50% softwood species (mainly loblolly, 
slash, sand, and pine) and 50% hardwood (mainly tupelo, gums, magnolia, and water 
oaks). At the time of this study, effluents received secondary treatment, which consisted 
of both anaerobic followed by aerobic biological degradation after a retention period of 
40 days. 

The objectives of this study were, first, to conduct a preliminary seasonal survey 
to examine the reproductive physiology of populations of Florida largemouth bass 
{Micropterus salmoides floridanus) sampled at increasing distances from a paper mill 
discharge area in relation to a reference site. For this part of the study, bass were sampled 
prior (September 1996) and during (February 1997) the spawning seasons. Since some 
reproductive alterations were observed in this preliminary survey, the second main 
objective was to further evaluate the possible impact(s) of environmental exposure to 
paper mill effluents on the reproductive physiology of this species by increasing the 
number of reproductive endpoints measured as well as the number of sites sampled. This 
second phase of the study was restricted to sampling during the reproductive season 
(March 1998). Parameters measured in these studies included body weights, lengths, 
condition factors, hepatic 7-ethoxyresorufin O-deethylase (EROD) activity, liver weights, 
gonad weights and histology, concentrations of vitellogenin and sex steroids 
(testosterone, 1 1-ke to testosterone, 17B-estradiol) and number and size of mature eggs in 
females. 



39 

Materials and Methods 

Sampling Sites and Fish Collection 

Field sampling was divided in two phases (see Table 2. 1 for a summary of the 
sampling methodology employed). The first phase included the sampling of 100 
largemouth bass (70 females and 30 males) during September 1996 (pre-spawning 
season) by electroshocking from four sites within the St. Johns River (Figure 2.1). 
Mainstream sites included a reference site located 40km upstream from the effluent 
discharge (Welaka), and three exposed sites located at increasing distance from the 
discharge (Palatka, Green Cove, and Julington Creek, at 3, 40, and 55km from the 
discharge, respectively). An additional 84 bass (36 females and 48 males) were sampled 
from the same sites during February 997 (spawning season). 

The second phase of the study was conducted during the spawning season (March) 
1998. A total of 61 females and 53 males were collected by electroshocking from six 
sites (-20 site) within the St. Johns River (mainstream) and its tributaries (small creeks) 
(Figure 2.1). Areas sampled included two tributary reference sites: Cedar Creek 
located approximately 25km downstream from the mill and Etonia Creek which is the 
primary water source for the mill and is located about 100-200 m upstream from the 
effluent discharge, and tributary exposed site Rice Creek, a small tributary stream 
(about 5 km in length) receiving the direct discharge from the mill. Fish were also 
sampled from two mainstream reference sites: Welaka and Dunn's Creek (the latter 
located 18km upstream from effluent discharge), and from mainstream exposed site 
Palatka, which receives the direct discharge from tributary Rice Creek. The estimated 






40 

paper mill effluent concentration in exposed sites Rice Creek and Palatka averages 60% 
and less than 10%, respectively (Georgia-Pacific Corporation, personal communication). 
However, water flow in Rice Creek is tidally influenced, so that during periods of low 
flow mill effluents can account for up to 90% of the total flow (Schell et al. 1993). 
Reference sites were matched to exposed sites in most characteristics, except presence of 
effluent. In order to minimize the variation in parameters measured in relation to timing 
of reproductive season, all fish within each site were collected within an average of four 
hours, and all sites were sampled in a 1-week period. Rice Creek was the only exception 
to this strict sampling protocol, where it was necessary to collect largemouth bass on 
three different occasions over a two-week period to achieve adequate numbers. 
Chemical Analysis from Fish Tissues 

With the objective of chemically characterizing some of the sites used in this 
study, fish tissues were collected and analyzed for up to 1 13 trace organics and 20 trace 
metal contaminants. For this analysis, livers from 5 females were collected from Welaka, 
Palatka, Green Cove, and Julington Creek during September 1996 and February 1997. 
Livers from the 1997 collection were pooled and run as one sample. Sample collections, 
laboratory analysis and quality control procedures were carried out as previously 
described (St. Johns Water Management District 1998). In brief, for determination of 
organics, samples were serially extracted using dichloromethane and then analyzed 
through gas chromatography/mass spectrometry (GC/MS) for determination of polycyclic 
aromatic hydrocarbons (PAHs) and phthalates or through GC/electron capture detection 
for analysis of polychlorinated biphenyls (PCBs), hexachlorocyclohexanes (BHCs), 
pesticides (dichlorodiphenyltrichloroethane and derivatives, DDTs), and other chlorinated 






41 

compounds. For metals, samples were digested with a mixture of nitric and hydrofluoric 

acids and concentrations were measured either by graphite furnace atomic absorption 

spectroscopy or inductively coupled plasma/mass spectrometry. Contamination data is 

reported on a dry weight basis, and was not corrected for lipid content, nor percent 

recoveries. 

Bleeding, Necropsies, and Age Determination 

Fish were weighed using a portable digital scale to the nearest O.lg and body 
length measured (total length, from the tip of the mouth to the tip of the tail) to the 
nearest millimeter. Condition factor was calculated as K = weight/length 3 x 100. Blood 
was collected in the field from the caudal vein using 3mL syringes and 1.5 inch, 20G 
needles. Blood samples were transferred to 5mL-heparinized vacutainers® and kept on 
ice until centrifugation for lOmin at 1,100 x g. Plasma was pipetted into 2mL cryotubes 
and stored at -80°C until analyzed. After bleeding, fish were euthanized with a blow to 
the head, and a complete necropsy performed. Gonads and livers were excised, weighed 
for the determination of organosomatic indices, and a section preserved in Notox® for 
histological evaluation as explained below. Fish collected during 1998 were decapitated 
for the removal of sagittal otoliths, which were used for the determination of age as 
described in Crawford et al. (1989). 
Reproductive Endpoints 

Analysis of sex steroid hormones 

Plasma samples from largemouth bass were analyzed for testosterone (only during 
1996/97 sampling), 11-ketotestosterone and 178-estradiol (all fish in the study) using a 



42 

radioimmunoassay (RIA) technique. The following is a description of the methodology 
for 176-estradiol and 11-ketotestosterone; the method for testosterone determination is 
similar. First, 50uL of plasma were extracted twice with 5mL of diethyl ether before RIA 
analysis. Samples were then analyzed in duplicate for both hormones and corrected for 
extraction efficiencies of 92 and 86 % for 178-estradiol and 11-ketotestosterone, 
respectively. Standard curves were prepared in buffer with known concentrations of 
radioinert 176-estradiol (ICN Biomedicals, Costa Mesa, CA, USA) or 11-ketotestosterone 
(Sigma Chemical, St. Louis, MO, USA) (1, 5, 10, 25, 50, 100, 250, 500, and l.OOOpg). 
The minimum concentration detectable was 6.4 pg/mL for 176-estradiol and 8.1 pg/mL 
for 1 1-ketotestosterone. All plasma samples were assayed in duplicate, and interassay 
variability was < 10% for each steroid. Values are reported as pg/mL of plasma. 

Cross-reactivities of 176-estradiol antiserum (produced and characterized by T. S. 
Gross, University of Florida) with other steroids were as follows: 11.2% for estrone, 1.7% 
for estriol, and < 1% for 17ct-estradiol and androstenedione. 11-ketotestosterone 
antiserum cross-reacted with: testosterone (9.7 %), a-dihydrotestosterone (3.7 %), and 
with androstenedione (< 1 %). A pooled sample (approximately 275pg of 176- 
estradiol/mL and 220pg of 1 1-ketotestosterone/mL) was assayed serially in 10, 20, 30, 40, 
and 50uL volumes (final volume of 50uL with charcoal-stripped plasma). The resulting 
inhibition curves were parallel to the respective standard curve. 
Analysis of vitellogenin 

Vitellogenin concentrations in plasma of largemouth bass were quantified by 
Direct Enzyme -Linked Immunosorbent Assay (ELISA). First, vitellogenin from 
largemouth bass was purified by anion exchange chromatography (LMB VTG 102396B), 



43 

and its protein concentration determined by the Bradford method (Bradford 1976) for use 
as a standard. The monoclonal antibody, Mab 3G2 Ascites 109 AB (produced by the 
Hybrydoma Core, University of Florida) was used in the ELISA assay. This antibody 
reacts with high specificity and sensitivity to largemouth bass vitellogenin, with little or 
no cross-reaction with other plasma proteins. 

Plasma samples were diluted from 1:200 (male samples) to 1:10,000 (female 
samples) in phosphate buffer saline azide (PBSZ 0.15 M NaCl, 10 nM phoshapte, 0.02% 
NaNj, pH 7.2) with aprotinin (10 KIU/mL), 50uL was added in triplicate to microtitre 
plate wells and incubated overnight at 4°C in a humidified chamber. Plates were then 
washed with PBSZ plus tween (PBSTZ, 0.05% Tween-20), blocked with 360 uL/well of 
blocking buffer (1% bovine serum albumin (BSA) and lOmM Tris BSTZ) for 2 hours at 
room temperature, and washed again with PBSTZ. Purified monoclonal antibody was 
diluted with blocking buffer with aprotinin to 3 ug/mL for male runs and to 0. 1 ug/mL for 
female runs, coated onto 96-well microtitre plates (50 uL/well), and stored overnight at 
4°C in a humified chamber. The next day plates were washed with Tris BSTZ, incubated 
with 50 uL/well polyclonal biotinylated goat mouse anti-vitellogenin IgG antibody (H + 
L) (Pierce, Rockford, IL, USA), diluted to 1:1,000 with blocking buffer, and incubated 
for 1 hour at room temperature. Plates were then washed with Tris BSTZ, and incubated 
with 50 uL/well of strep-avidin-alkaline phosphatase, diluted to 1:1,000 with blocking 
buffer, for 1 hour at room temperature. After a final wash with Tris BSTZ, 100 uL/well 
of p-nitro phenyl phosphate in carbonate buffer (pH 9.6) was added to each well and 
incubated at room temperature in the dark for 30 minutes. The intensity of yellow color 
that developed was quantified at 405nm with and automated ELISA reader (Spectra Max 



44 

250, Molecular Devices, Sunnyvale, CA, USA). Vitellogenin concentrations were 
calculated from standard curves after subtracting the small blank value (around 0.2 A405 
nm ) of a nonspecific color reaction with male control plasma. 

Standard curves were constructed by adding serial dilutions of purified 
largemouth bass vitellogenin (0 mg/mL to 0.001 mg/mL) to male control plasma and 
processed the same way as samples. Male control plasma was made from a pool of 
plasma from fish collected at an uncontaminated site, which was shown by Direct ELISA 
and Western Blot analysis to have no vitellogenin. Each assay was run with a positive 
control with a known vitellogenin concentration, to test for interassay and intra-assay 
variation. Samples were rerun if the coefficient of variation between triplicates exceeded 
10%. Standard curves fit by quadratic regression were used to calculate vitellogenin 
concentration, with R values usually between 0.95 and 0.99. The minimum 
concentration detectable in this assay is of 0.001 mg/mL. Values are reported as mg/mL 
of plasma. 
Gonadosomatic (GSIs) and hepatosomatic (HSIs) indices 

Gonads and livers (without gall bladder) were excised from each fish and weighed 
using a portable scale to the nearest O.Olg. Somatic indices were calculated by dividing 
the weight of the organ by the weight of the fish and multiplying the resulting number by 
100. 
Number and size of mature eggs 

The total number of mature eggs was estimated in bass sampled during 1998 by 
collecting a subsample of follicles (approximate wet weight of lOOmg) along a mid- 
section of one of the ovaries. The sample was preserved in Notox ® and the number of 



45 

mature eggs (defined as tan- yellow to brown-yellow eggs of over 0.6mm in diameter) 
counted under a dissecting scope. To obtain the total number of mature eggs in both 
ovaries, the number of mature eggs in the subsample was multiplied by the weight of both 
ovaries and the resulting number divided by the weight of the subsample. The mean size 
or diameter of mature eggs was calculated by lining 10 eggs on a ruler under a dissecting 
microscope and dividing the resulting number by 10. This procedure was done twice, so 
that 20 eggs were measured from each sample of follicles. 
Histopathology 

During the 1998 study, samples of gonads were collected and preserved in 
Notox® for histological evaluation. Testes were cut longitudinally and ovaries were cut 
transversally. Tissue samples were then embedded in paraffin, sectioned at 5um, 
mounted on glass slides, air dried and stained with Mayer's hematoxylin and eosin 
(H&E). Ovaries were classified into four stages of sexual maturation: undeveloped 
(stage 1, mostly primary oocytes at various stages of previtellogenic growth); 
previtellogenic (stage 2, primary and secondary oocytes, no vitellogenic oocytes); early 
vitellogenic (stage 3, some vitellogenic oocytes of different sizes, with few to moderate 
amount of vitelline granules, and few to no fully developed eggs); and late vitellogenic 
(stage 4, most of the oocytes contained numerous vitelline granules). In addition, the 
number of atretic follicles was counted in each histologic section of ovaries. Testes were 
classified into three stages of sexual maturation: low to no spermatogenic activity (stage 
1, thin germinal epithelium, scattered spermatogenic activity); moderate spermatogenic 
activity (stage 2, thick germinal epithelium, diffuse to moderate proliferation and 



46 

maturation of sperm); and high spermatogenic activity (stage 3, thick germinal 
epithelium, high proliferation and maturation of sperm). 
Liver EROD Activity 

For this assay, buffers, substrates, and cofactors were purchased from Sigma 
Chemical, St. Louis, MO, USA. Snap frozen livers from fish collected during 1998 were 
cut with a hammer and chisel so as to collect a total of approximately 250mg of tissue. 
Samples were homogenized with 3 volumes (v/w) of homogenizing buffer consisting of 
lOmM TRIS (pH 7.4), 250mM sucrose, ImM EDTA, 0.2mM dithiothrietol, and O.lmM 
phenylmethylsulfonyl fluoride. Samples were homogenized for approximately lOsec, and 
the homogenates were centrifuged at 8000 x g for 10 min. The 10,000g supernatant (S-9 
fractions) containing microsomal enzymes was isolated by centrifugation at 12,000 x g 
for 20 min and stored at -80°C until analyzed. S-9 proteins were assayed by the BioRad 
protein assay kit (Richmond, CA, USA) using bovine serum albumin as a standard. Liver 
samples were kept ice-cold (4°C) throughout. Hepatic EROD kinetic activity was 
measured in triplicate in the S-9 fractions using a Spectromax Fmax 96 fluorescent 
microplate reader at an excitation wavelength of 544nm and emission at 590nm. For this 
reaction, 5uL of enzyme (S-9) were mixed with 195uL assay buffer (0.1 m NaP04, pH 
7.8) and 5uL substrate (lOOuM ethoxyresorufin in methanol). The reaction was started by 
adding 5uL NADPH, and the fluorescence change recorded for 2 min at 30°C. Negative 
controls were run in the absence of enzyme and positive controls in the presence of rat 
microsomes. Reaction linearity was demonstrated over the course of the reactions. A 
resorufin standard curve (0, 1, 2.5, 5, 10, and 20pmol) was run for each set of samples. 
EROD reaction rates were determined by dividing the rate of change in fluorescence per 






47 

minute by the slope of the resorufin standard curve. Results are expressed as pmol of 
resorufin formed/min/mg microsomal protein. 
Statistical Analyses 

Pairwise comparisons were conducted using a two-way analysis of covariance 
(ANCOVA) (PROC GLM, SAS Institute 1988) within sexes and years (1996-97 and 
1998) to test for differences in the dependent variables between sites. Data sets that did 
not meet the criteria of normality and homogeneity of variance (PROC UNIVARIATE) 
were log or arcsin transformed. For the 1996-97 data set, season (spawning or non- 
spawning) was used as the second cofactor and body weight was used as the covariate, 
whereas for the 1998 fish, type of stream (tributary or mainstream) was used as the 
second cofactor and age was used as the covariate. For the 1996-97 data, exposed sites 
Palatka, Green Cove, and Julington Creek were compared against the reference Welaka, 
while for the 1998 study, exposed sites Palatka and Rice Creek were compared to the 
reference sites Welaka and Dunn's Creek and Cedar and Etonia Creeks, respectively. If 
the ANCOVA showed a significant site effect, a Dunnett's multiple comparison test was 
used to examine which exposed site(s) differed from the reference. Regressions between 
gonad and liver weight to body weight were compared among reference and exposed sites 
after combining data from spawning seasons 1997 and 1998. In addition, regressions of 
liver EROD activity and several reproductive parameters measured in females collected 
during 1998 are also presented. The frequency distributions of different gonadal 
developmental stages were compared between sites using a X Test (PROC FREQ). For 
purposes of statistical comparisons, ovaries and testes were classified as either low to 



48 

moderate (stages 1 and 2 for both sexes) or high gametogenesis (stages 3 for males, and 3 
and 4 for females). Statistical significance was assessed at p < 0.05. 

Results 

Chemical Analysis from Fish Tissues 

A summary of fish chemical data is presented in Table 2.2. With the exception of 
BHCs, the sum of organics measured in fish tissues appeared highest in bass collected 
from exposed site Palatka when compared to fish from reference site Welaka (increases 
ranged from 1.5 to 7-fold). There was also an overall trend for a decline in organic 
chemicals in fish from exposed sites Green Cove and Julington Creek in relation to fish 
from Palatka, with several groups being lower (low molecular PAHs and BHCs) or 
comparable (chlorinated benzenes and other chlorinated pesticides) to values found in 
reference fish (Table 2.2). Metals were more variable across sites, with highest mean 
concentrations mainly found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or 
Welaka (Cd, Hg, Pb, Se, Tn). 
Physiological and Reproductive Endpoints 

1996-97 field study 

A summary of several physiological and reproductive parameters measured from 
largemouth bass sampled along the St. Johns River during September 1996 (pre- 
spawning) and February 1997 (spawning) is presented in Table 2.3. Females from Green 
Cove and Julington Creek were smaller and lighter when compared to females from the 
reference site (Welaka), whereas males did not differ between sites. For both sexes, body 
weights, lengths and condition factor did not differ across seasons. There were 



49 

differences in gonad weights and GSIs across sites. Palatka and Julington Creek females 
had gonad weights and GSIs that were approximately half of those reported from Welaka. 
Males from Palatka and Julington Creek had lower and higher gonad weights and GSIs, 
respectively when compared to males from the reference site. 

For both sexes, plasma concentrations of testosterone were not affected by site, 
but decreased from September to February (from a mean of 397 to 338 pg/mL in females, 
and from 404 to 207 pg/mL in males) (Figure 2.2). Females from Green Cove had almost 
twice the concentration of 1 1-ketotestosterone when compared to the reference (for both 
sampling periods), and this hormone increased from an average of 267 pg/mL in pre- 
spawning bass to 448 pg/mL in fish sampled during the spawning season (Figure 2.3). 
Pre-spawning males from Palatka and Julington Creek had slightly lower concentrations 
of 1 1-ketotestosterone when compared to the reference stream, but when sampled during 
the spawning season Welaka males had concentrations of 1 1-ketotestosterone that were 
over twice of that found in males from exposed sites (Figure 2.3). There were seasonal 
changes in the concentration of 1 1-ketotestosterone in males from all sites except Green 
Cove (increased from a mean of 319 pg/mL in September to 628 pg/mL in February). For 
both sampling periods, 178-estradiol was lower in Palatka females, and the concentration 
of this hormone increased from September to February (mean = 325 to 927 pg/mL) 
(Figure 2.4). In contrast, plasma concentrations of 178-estradiol increased in pre- 
spawned males from all sites and in spawned males from exposed Palatka and Green 
Cove sites in relation to the reference. Seasonal effects were observed only in males 
collected from Palatka and Julington Creek (Figure 2.4). During the spawning season, 
the ratio of 178-estradiol to 1 1-ketotestosterone (E/l 1-KT) decreased from a mean of 4.2 



50 

in females from the reference site to 1.8 in bass from exposed sites (Figure 2.5). In 
contrast, E/l 1-KT ratios increased in males from all sites (except for Julington Creek 
males sampled during February) from 0.36 to 1.22 due to a decline in 1 1-ketotestosterone 
and an increase in 178-estradiol. Vitellogenin concentrations were approximately 17 
times lower in spawning females from Palatka and Green Cove in relation to the 
reference (mean = 0.42 and 7.0 mg/mL, respectively) (Figure 2.6). Although Julington 
Creek females had about half the concentration of this protein when compared to bass 
from Welaka, this difference was not significant. Seasonal changes in vitellogenin 
concentrations were observed only in females sampled from Welaka and Julington Creek 
(from September to February it increased over 1000-fold from 0.005 to 5.42 mg/mL). 
Although male bass had comparable concentrations of vitellogenin across sites, as with 
females, plasma concentrations of this protein increased from a mean of 0.003 mg/mL in 
males sampled during September, to a mean of 0.006 mg/mL in males collected during 
February (Figure 2.6). 
1998 field study 

A summary of several physiological and reproductive parameters measured from 
largemouth bass sampled along the St. Johns River during March 1998 is presented in 
Table 2.4. Age was affected only at tributary Rice Creek, where females were 
significantly younger when compared to the reference tributary sites. There were no other 
differences in the remaining parameters among reference and exposed sites. Females 
sampled from tributaries, however, were older, heavier and longer than females collected 
from mainstream sites (4.1 vs. 3.4 years; 1041 vs. 738g; and 41 vs. 37cm, respectively). 
Although liver weights were higher in females collected from tributary streams when 



51 

compared to mainstream values (12.1 vs. 8.3g), GSIs were lower in these females (3.4 vs. 
2.4%). Males from tributaries, on the other hand, had higher condition factors, and higher 
HSIs and liver weights in relation to mainstream stations (1.6 vs. 1.4; 1.4 vs. 0.91%, and 
8.4 vs. 5.6g, respectively), but lower GSIs (0.35 vs. 0.47%). 

Female largemouth bass sampled from a small creek receiving the direct discharge 
from the mill (Rice Creek) had a 5-fold increase in EROD activity compared to bass 
sampled from reference streams (Figure 2.7). Males had EROD activities that were about 
twice as high as females (mean = 6.7 vs. 3.0 pmol resorufin/mg/min for males and 
females, respectively), but that did not differ across sites. Vitellogenin concentrations 
were not affected by site of collection, and averaged 0.66 mg/mL in females and 0.08 
mg/mL in males (Figure 2.7). Concentrations of 11-ketotestosterone and 178-estradiol in 
females from exposed sites were about half and 1/3, respectively of those from the 
reference station (Figure 2.8). In males, 178-estradiol did not change between exposed 
and reference sites, but 1 1-ketotestosterone decreased in tributaries and mainstream 
exposed sites in relation to controls. E/l 1-KT ratios were decreased and increased in 
females and males, respectively from exposed stations (Figure 2.9). Males from exposed 
tributaries also had higher E/l 1-KT ratios when compared to males sampled from 
exposed mainstream sites (2.2 vs. 0.65). Although females from Rice Creek tended to 
produce fewer and smaller eggs when compared to females from reference Cedar and 
Etonia Creeks, this difference was not significant (Figure 2.10). In contrast, and despite 
the decline in sex steroid concentrations observed in females from Palatka, this group 
produced almost as twice as many eggs in relation to bass from reference Welaka and 
Dunn's Creek. The size of these eggs, however, was similar between the two types of 






52 

mainstream sites (Figure 2.10). Overall, females collected from tributary sites had lower 
fecundities and egg sizes than females from mainstream sites (mean = 14,856 vs. 17,017 
eggs, and mean = 0.75 vs. 0.99mm). Differences in reproductive parameters between 
reference and exposed sites occurred despite the fact that there were no differences in 
reproductive development as measured in histological sections of ovaries and testes 
(Figure 2.1 1). There was, however, an effect of type of stream on ovarian development, 
with a higher proportion of females from mainstream sites having ovaries with a high 
degree of oogenesis when compared to tributary sites (62 vs. 38%). The number of 
atretic follicles/histological section did not differ among reference and exposed sites 
(Figure 2.12), but was significantly increased in females from tributary sites when 
compared to bass from mainstream areas (mean = 9.2 vs. 3.5). In addition, the level of 
ovarian development was negatively correlated to the number of atretic follicles (R = - 
0.55, p = 0.0001). Regression analyses between reproductive parameters and liver EROD 
activity in female largemouth bass from exposed tributary and mainstream sites is 
presented in Figure 2.13. Vitellogenin, logarithm of gonad weight, GSI, fecundity, and 
egg size were inversely related to liver EROD activity. 
All years of field studies 

Regression lines between body lengths and organ weights versus body weight for 
spawning bass from reference and exposed sites collected during 1997 and 1998 are 
presented in Figure 2.14. When all spawning fish were combined, there were no 
differences among sites in the linear regressions relating body weight to body length and 
gonad and liver weight (i.e. all slopes were parallel). The size distribution of females and 
males during the spawning sample (1997 and 1998 combined) was shifted towards fish 



53 

that ranged in length from 31 to 49cm (91% females and 93% males fell in this size 
range), and was similar among reference and exposed sites (Figure 2.15). 

Discussion 

Although one of the main objectives of this study was to evaluate potential 
impacts of BKME on populations of largemouth bass, during the first year of study fish 
were also sampled at considerable distances from the mill discharge (Green Cove and 
Julington Creek located at 40 and 55km from the mill, respectively). Female bass 
sampled from Palatka (closest to the mill) and Green Cove had lower concentrations of 
176-estradiol, vitellogenin, lower E/ll-KT ratios, and lower GSIs in relation to the 
reference site. Females from Julington Creek, however, only showed lower E/l 1-KT 
ratios. Males from Palatka and Green Cove showed higher increases in 176-estradiol and 
in E/l 1-KT ratios, but comparable declines in 1 1-ketotestosterone in relation to males 
from Julington Creek, and GSIs were decreased only in Palatka males. These results 
indicate a geographical trend in reproductive effects, with decreasing changes as the St. 
Johns River flows north. In addition, the presence of reproductive alterations in fish 
sampled at a considerable distance from the mill discharge would suggest exposure to 
chemicals other than BKME. In this respect, there is considerable evidence showing 
endocrine alterations in fish due to exposure to different groups of chemicals not 
necessarily related to BKME (such as metals, halogenated aromatic hydrocarbons, and 
chlorinated hydrocarbons) (Kime 1995, Heath 1995b, Giesy and Snyder 1998). 

Except for higher values of some organics and metals in fish tissues from Palatka 
and Julington Creek, the chemical data available was not powerful enough to detect clear 






54 

trends across sites. This was probably related to the small number of samples analyzed, 
which resulted in a high degree of variation. Although no fish tissues from the site 
closest to the discharge (Rice Creek) were available for chemical analysis in this study, 
there is indication that this stream is being impacted as a result of effluent discharge. 
Schell et al. (1993) reported up to 52.8 ppt of 2,3,7,8-tetrachlorodibenzo-p-dioxin 
(TCDD) in sediments collected from Rice Creek, with significant declines in the 
concentration of this chemical at the confluence with the St. Johns River (6.8 ppt at Site 
3). These authors also reported concentrations of TCDD in liver and gonads of 
largemouth bass (range 1.8 - 8.8), bowfin, Amia calva (1 1.2 - 46.1), and brown bullhead 
catfish, Ictalurus nebulosus (1.8 - 2.8) collected from Rice Creek. Another indication of 
the presence of effluent chemicals in Rice Creek comes from the work by Quinn (2000) 
who found a distinct gradient in the waterborne concentrations of three resin acids 
(dehydroabietic, abietic, and isopimaric acids) with highest concentrations at the site of 
effluent discharge and non-detectable levels at the confluence of Rice Creek with the St. 
Johns River (Palatka site). 

Measurements of EROD activity have been widely used as a biomarker for 
exposure of fish to several groups of chemicals, including polychlorinated dibenzo-p- 
dioxins (PCDDs) and dibenzofurans (PCDFs), PCBs, PAHs, pesticides, metals, and 
natural biogenic substances. Because BKME are known to contain EROD-inducing 
compounds, this biomarker has played a major role in the study of fate and biological 
effects of paper mill effluent discharges. In general, researchers have reported low EROD 
activities in fish from reference sites, with significant increases in areas close to pulp mill 
outfalls (Forlin et al. 1985, Lindstrom-Seppa and Oikari 1989, Courtenay et al. 1993, 



55 

Bankey et al. 1994, Soimasuo et al. 1995b). Until recently, it was believed that the main 
inducers in mill effluents were chlorinated persistent compounds (such as PCDDs and 
PCDFs) (Hodson 1996). However, new evidence suggests that enzymatic EROD 
induction also occurs in fish exposed to unbleached effluents, and that the compound(s) 
responsible for such induction are not of the highly hydrophobic chlorinated type, but 
rather of the moderately hydrophobic planar PAH-type form present as natural 
components of wood, and readily metabolized by fish (Hodson 1996). 

In the present study, EROD activity was induced in females from the site closest 
to the mill outfall (Rice Creek) when compared to the reference (8.4 vs. 1.7 
pmol/min/mg, about a 5-fold induction). There was also a gradient of induction from 
Rice Creek to its confluence with the St. Johns River (a distance of only 3km), with 
females from this latter site having EROD activities that were comparable to values from 
reference areas (1.2 pmol/min/mg). This rapid fall in mixed-function oxygenase (MFO) 
activity would suggest that compounds capable of causing enzyme induction are present 
in high enough concentrations only in water and/or sediments from Rice Creek, and that 
by the time they reach the St. Johns River they are diluted enough for EROD activities to 
fall to background levels. There is also evidence for a decline in total organic content 
(TOC) in sediments from mainstream Palatka in relation to Rice Creek (from 36 to 25 
mg/g) (Schell et al. 1993). This decline in TOC could imply that, upon entering this 
creek, lipophilic organic contaminants would associate at higher levels with organic-rich 
sediments present in Rice Creek with lower associations in sediments from the 
mainstream. 



56 

EROD activities in males were more variable across sites, and although they 
appeared higher in bass sampled from Rice Creek and Palatka (average of 9.1 
pmol/min/mg) when compared to males from reference sites (average of 5.4 
pmol/min/mg), these differences were not statistically significant. The uniformity of 
hepatic MFO induction in male largemouth bass from reference and exposed sites would 
suggest a higher degree of mobility compared to females. Information on the range of 
movement of largemouth bass is restricted to the work by Snyder et al. (1986) who 
reported that 38% of the bass marked and released in the lower St. Johns River were 
recaptured in the same are as tagged, and of the remaining 62%, 44% had moved a 
distance of less than 2km. Unfortunately, the study did not evaluate differences in range 
between sexes. 

The degree of EROD activity was different between female and male largemouth 
bass, with males having approximately twice the activities seen in females. This 
differential activity level could be reflecting differential body burdens of organic 
contaminants due to sex differences in lipid concentrations and/or food habits. A more 
plausible explanation for sex differences in enzyme activity, however, is that EROD 
levels are known to be reduced or even eliminated in female fish during reproduction 
(Elskus et al. 1989, Larsen et al. 1992, Stegeman and Hahn 1994). Although the exact 
mechanism of this regulation remains unclear, the suppression is thought to be related to 
higher increases in 176-estradiol concentrations in spawning females in relation to males. 
If a similar pattern occurs in spawning largemouth bass, it is likely that we 
underestimated EROD basal activities in this study. In this respect, EROD basal 
activities of adult bass in this study are much lower than the values reported in immature 



57 

largemouth bass exposed to up to 8% BKME for 263 days (which resulted in up to 800 
pmol/min/mg of activity) (Bankey et al. 1994), although the induction levels were similar 
(up to six-fold). This large difference in absolute EROD values is probably related to 
having worked with adult spawning bass as opposed to juvenile fish. On the other hand, 
induction levels in females from Rice Creek are comparable to values reported by Haasch 
et al. (1993) in bass (age not reported) exposed in the laboratory to B-naphthoflavone for 
up to 4 days, and to caged bass exposed to waters from a PAH and PCB-contaminated 
river for up to 7 days (overall inductions of 4.4 and 6, respectively). 

An area of intense research has been to try to relate increases in EROD activity 
with changes in physiological and reproductive endpoints. Studies on the effects of paper 
mill effluents on fish have provided most of the information available on this subject. 
The overall conclusion from these studies is that there appears to be no clear relationship 
between decreased titers of steroid hormones and other reproductive alterations and 
increased hepatic EROD induction (Munkittrick et al. 1992b, 1994, Gagnon et al. 1994a, 
1994b, Soimasuo et al. 1998). Preliminary results from the present study, however, 
indicate a significant inverse relationship between many reproductive endpoints measured 
in female bass (vitellogenin, GSI, log gonad weight, fecundity and egg size) and EROD 
activity (see Figure 2.13). The consistency of this negative relationship is suggestive of 
an association between antiestrogenic effects and EROD induction in largemouth bass. 
English sole (Parophrys vetulus) exposed naturally to Puget Sound sediments 
contaminated with PCBs and PAHs, also showed significant correlations between 
chemical exposure, MFO induction, and reduced concentrations of plasma 176-estradiol 
(Johnson et al. 1988). In this respect, mechanistic in vitro studies have shown a negative 



58 

relationship between a compound's antiestrogenicity and it's ability to induce cytochrome 
P450-dependant monooxygenase (CYP1 A) proteins (Anderson et al. 1996). These 
authors also reported depression of estrogen receptor binding capacity in relation to 
increased EROD activity in juvenile rainbow trout (Oncorhynchus mykiss) injected with 
50 mg/kg of P-naphthoflavone. These findings indicate alterations in the affinity of 17B- 
estradiol for the estrogen receptor, probably due to Ah-receptor mediated changes in the 
phosphorylation state of the estrogen receptor (Anderson et al. 1996). Since CYP1 A in 
fish does not participate in the catabolism of 17B-estradiol, it is unlikely that increases in 
EROD activities are related to increases in the oxidative metabolism of this hormone 
(Snowberger and Stegeman 1987). It is clear that additional studies are needed for a 
better understanding on the involvement of EROD enzymatic activity on endocrine 
modulation and its potential deleterious effects on fish reproduction. 

Many studies on the effects of paper mill effluents, report a concomitant increase 
in HSIs in fish with high EROD activities (Larsson et al. 1988, Munkittrick et al. 1992a, 
1994, Bankey et al. 1994, Huuskonen and Lindstrom-Seppa 1995). Although we did 
observe an increase in EROD activity in at least some of the fish analyzed, this induction 
was not associated with increases in liver size. The absence of an increase in HSIs in the 
present study could have been related to the timing of fish sampling. Bass were collected 
during the reproductive season (March), and it is well known that the weight of the liver 
in this species undergoes seasonal variations, with highest values in winter and spring 
(December-April) and lowest values in the summer months (Adams and McLean 1985). 
Thus, any increases in liver weight due to enhanced activity of xenobiotic 
biotransformation enzymes would have been masked by the physiological increases that 



59 

are associated with reproductive status (this is particularly true in the case of females due 
to increases in the synthesis of vitellogenin). Lack of increases in liver weight after 
exposures to pulp mill effluents have also been reported in longnose sucker (Catostomus 
catostomus) (Munkittrick et al. 1992), trout-perch (Percopsis omiscomaycus) (Gibbons et 
al. 1998), and whitefish {Coregonus muksuri) (Lindstrbm-Seppa and Oikari 1989). 

There were similarities, but also differences in the reproductive responses of bass 
from the 1996/97 and 1998 studies (Table 2.5). For both years of study, female bass 
collected from reference sites had higher concentrations of 178-estradiol (between 42 and 
64% higher) whereas males from these sites had higher concentrations of 11- 
ketotestosterone in relation to fish from exposed sites (between 46 and 67% higher). 
Concentrations of plasma testosterone appeared less sensitive than those of 176-estradiol 
and 1 1-ketotestosterone, with changes only across seasons but not sites. Year differences 
included declines in vitellogenin in females and in GSIs (both sexes), and increases in 
178-estradiol in males from exposed sites only during the February 1997 sampling. In 
addition, concentrations of 1 1-ketotestosterone in spawning females from exposed sites 
behaved oppositely across years, with increases during 1997 and declines in 1998 (see 
Table 2.5). 

Because bass were collected from both mainstream and tributary streams during 
1998 but only from the former in 1997, comparisons among years are most appropriate if 
restricted to responses of mainstream spawning fish. An explanation for the differences 
in reproductive responses between years could be related to the timing of sampling. 
During 1998 mainstream bass were sampled a month later in the reproductive season 
(March as opposed to February in 1997), which resulted in higher GSIs and plasma 



60 

concentration of sex steroids in fish from both sexes in relation to 1997. The only 
reproductive parameter that did not follow this same trend was vitellogenin in females, 
which was lower in 1998. This is not surprising since plasma concentrations of this 
protein are known to decline by over 50% from February to March (from about 4 to 2 
mg/mL) in female bass (Timothy Gross and Nacy Denslow, unpublished data). It is 
possible then, that in order to detect small differences in GSIs and vitellogenin between 
clean and contaminated streams bass need to be sampled earlier during the reproductive 
season. In addition, because of the high dynamism of the hydric system under study, it is 
expected that year differences in chemical composition may occur through time (Durell et 
al. 1998). This coupled with the fact that largemoufh bass are likely to move some 
distance from year to year (Snyder et al. 1986) could result in a differential degree of 
exposure, and thus on differences on the physiological responses being measured. 

Despite the differential response in sex steroids among years, there was an overall 
similar decline (31 - 58%) and increase (61 - 66%) in E/l 1-KT ratios in females and 
males, respectively for both years of study. Throughout this study, E/l 1-KT ratios were 
above 3 (4.2 in 1997 and 3.4 in 1998) in plasma of spawning female bass from reference 
sites and below 0.5 in males (0.3 in 1997 and 0.4 in 1998). E/l 1-KT ratios above 3 are 
expected in healthy reproductively active female bass because of a predominance in 17B- 
estradiol over 1 1-ketotestosterone. Conversely, low E/l 1-KT ratios in males indicate a 
higher proportion of 1 1-ketotestosterone over 17B-estradiol. An imbalance between the 
concentration of these two sex steroids, as that observed in fish collected from exposed 
streams (E/ll-KT ratios of 1.8 and 2.4 in females from 1997 and 1998, and of 0.9 and 1.3 
in males from 1997 and 1998) may be indicative of endocrine disruption. 






61 

In the present study, season and type of stream affected some of the reproductive 
parameters measured. From the 1996/97 seasonal study it was apparent that although 
changes in sex steroids between sites were observed as early as 5 months prior to 
spawning (September), these were most evident in spawning fish (February). On the 
other hand, results from the 1998 study would suggest that fish collected from tributary 
streams were at a slighter earlier reproductive stage than animals from the mainstream. 
For example, even though females from tributary streams tended to be older and heavier, 
they also had lower GSIs, fecundities, and egg sizes in relation to females from 
mainstream sites. Similarly, males from tributary streams had higher condition factors, 
but lower GSIs. In both sexes, liver weights were also increased in bass collected from 
tributary stations, which would suggest that at the time of sampling these fish had 
allocated less body reserves into reproduction in comparison to mainstream bass. These 
reproductive differences were corroborated histologically in females, with tributary fish 
having a lower frequency of ovaries with high degree of oogenesis and an increase in the 
number of atretic follicles. It is worth noting, however, that despite the reproductive 
changes just mentioned concentrations of sex steroids and vitellogenin did not differ 
among bass from mainstream and tributary streams in either sex. For future studies then, 
it would be of importance to take into consideration the seasonal fluctuations in 
reproductive parameters, as well as the effect of type of stream on the reproductive 
physiology of this species of fish. 

There was a lack of consistency on the effects of environmental contaminants on 
GSIs. During 1997, GSIs were decreased in females from exposed sites Palatka and 
Green Cove, but not in females from exposed Julington Creek. Although the decline in 



62 

176-estradiol was comparable across females from exposed sites (42% decline overall), 
females from Julington Creek (site furthest away from the mill discharge) had 
vitellogenin concentrations that were not significantly different in relation to females 
from the reference site. A higher concentration of vitellogenin in females from this site 
could explain a lack of a decline in GSIs. During this same year, GSIs in males were 
lowered only in fish collected from the site closest to the mill discharge (Palatka), despite 
a similar decline in 11-ketotestosterone across exposed sites (46% decline overall). 
Similarly to what was observed with females, however, males from Julington Creek had 
increased GSIs, which could be related to a lack of an increase in 17B-estradiol in these 
males in relation to males from Palatka and Green Cove. In the 1998 field study, a 
significant decrease (42 to 67%) in reproductive hormones was observed in females and 
males from exposed sites. Gonadosomatic indices, however, were either slightly 
decreased in the case of males (19%, although not statistically significant) or not affected 
at all in the case of females. Despite some declines in GSIs in fish from exposed sites 
during the spawning season of 1997, when data from both years was combined, there was 
a clear lack of a relationship between GSIs and site of collection (Figure 2.14). These 
results suggest that variations in gonad weight are probably more related to variations in 
local environmental conditions rather than to contaminant exposure, and/or that higher 
declines in sex steroids and vitellogenin are necessary before declines in GSIs are 
observed. Several studies have reported declines in GSIs in fish exposed to BKME 
(Larsson et al. 1988, Munkittrick et al. 1991, 1992a, 1994, Gagnon et al. 1994b, Gibbons 
et al. 1998). However, there is also evidence to suggest that decreases in gonadal size in 
response to declines in sex steroids may not always occur after exposures of fish to 






63 



BKME (McMaster et al. 1996b), which would indicate differences in reproductive 
responsiveness to contaminant exposure across species. 

The present study is one of a few to evaluate changes in vitellogenin 
concentrations in relation to exposure to paper mill effluents. Although there was a 
decline in the plasma concentration of this protein in females from exposed sites when 
compared to reference streams during both years of study (94 and 56% declines for 1997 
and 1998, respectively), this decline was significant only in 1997. Higher declines in 
vitellogenin in females sampled during February 1997 could explain the concomitant 
decrease in GSIs and the lack of such a decline in March 1998. Declines in vitellogenin 
and 176-estradiol in females from contaminated streams would suggest exposure to 
antiestrogenic compounds. 

The presence of vitellogenin in plasma of male largemouth was a consistent 
finding in this study. The concentrations of vitellogenin found in males, however, were 
an average of about 1/12 of those found in females, and although increased from 
September to February, its high variability precluded the detection of any differences 
between exposed and reference sites. Finding detectable concentrations of vitellogenin in 
plasma of male fish has generally been considered as a sign of endocrine disruption 
associated with exposure to estrogenic compounds (Sumpter and Jobling 1995). Plant 
sterols, such as P-sitosterol, which are commonly found in pulp mill effluents, are 
estrogenic compounds known to bind in vitro to rainbow trout hepatic estrogen receptors 
(Tremblay and Van Der Kraak 1998) and induce vitellogenin synthesis in male goldfish 
(Carassius carassius) (MacLatchy and Van der Kraak 1995). Although we can not rule 
out the possibility that male bass sampled closest to the mill discharge could have been 



64 

exposed to the estrogenic effects of plant sterols, several reports have documented low 
background concentrations of vitellogenin in different species of male fish, and have 
regarded such concentrations as physiologically normal (Copeland and Thomas 1988, 
Ding et al. 1989). The results from the present study, consistently showing low 
concentrations of vitellogenin in male largemouth bass sampled from both clean and 
contaminated streams, further supports this hypothesis, and cautions the use of just the 
presence of this protein in plasma of males as a definitive sign of endocrine disruption. 

During the 1998 study, gonads were evaluated histologically to ensure that 
observed differences in concentrations of sex steroids and vitellogenin between fish from 
exposed and reference sites were not caused by different stages of sexual maturity. 
Within each type of stream, the results showed similar stages of ovarian and testicular 
development among bass collected from all three sites. In addition, the similarity in the 
number of atretic follicles in ovaries from females from clean and contaminated streams 
and the absence of any noticeable lesions in the testes examined would suggest that 
alterations in sex steroid concentrations in bass from exposed sites (declines in 176- 
estradiol and 11-ketotestosterone in females and males, respectively) were probably not 
enough to cause damage to gonadal tissue. 

A rather unexpected finding in this study was that females from Palatka had 
ovaries that contained more eggs in comparison to females from the reference sites and 
females from Rice Creek. These eggs were also larger, but only when compared to fish 
from Rice Creek. Females sampled from the Palatka site also had larger gonads (4% 
body weight vs. 3% in females from reference sites), although these differences were not 
statistically significant. These results occurred despite the fact that Palatka females had 



65 

less than half the concentrations of 176-estradiol in relation to females from the reference 
sites (611 vs. 1513 pg/mL). Increases in fecundity that were coupled with declines in 
plasma sex steroids have also been reported in lake whitefish (Coregonus clupeaformis) 
females exposed to BKME (Munkittrick et al. 1992a). In contrast to what was observed 
in the present study, however, lake whitefish females also had lower gonad weights, 
which resulted in smaller egg sizes in comparison to reference females. White suckers 
from metal-contaminated sites have also shown increased fecundities (Munkittrick and 
Dixon 1989), but these females also had reduced spawning and the hatching rate was 
decreased because of egg shell-thinning problems. Increases in fecundity and fertility 
have also been observed in certain fish populations, and have been interpreted as an 
adaptation to high levels of pollution (by securing the production of more offspring these 
populations will eventually lead to the formation of a more pollutant resistant strain of the 
species) (Kime 1995). At this point, the origin of the increased fecundities in females 
from Palatka, as well as the population-level effects that could be associated with such a 
change remain unknown. 

One of the most consistent findings in studies that have focused on the effects of 
BKME on reproductive parameters of fish is a decline in the concentration of sex steroids 
in plasma of exposed animals. BKME-exposed white suckers from Jackfish Bay, Lake 
Superior show decreased concentrations of several sex steroid hormones (testosterone, 
11-ketotestosterone 176-estradiol, and 17, 20p-dihydroxy-4-pregnen-3-one) (Portt et al. 
1991, Mc Master et al. 1995, 1996b). Declines in steroid concentrations have also been 
documented in longnose sucker and lake whitefish from Jackfish Bay (Munkittrick et al. 
1992a, McMaster et al. 1996b), in white sucker at other mills (Hodson et al. 1992, 



66 

Munkittrick et al. 1994, Gagnon et al. 1994a), and in other fish species sampled 
elsewhere (Adams et al. 1992, McMaster et al. 1996b). The consequences of these 
similar endocrine alterations to whole animal reproductive fitness and population 
dynamics, however, have varied greatly between species. For example, longnose sucker 
exposed to BKME show no organism responses other than an altered age distribution, 
whereas white sucker and lake whitefish show decreased gonadal sizes, secondary sexual 
characteristics, and egg sizes, and increased age to maturity (McMaster et al. 1996b). In a 
review of whole organism responses of fish exposed to different kinds of mill effluents 
(including unbleached pulps), 48% of the populations studied had increased condition 
factors, 80% showed increased age to sexual maturation, and reduced gonadal size was 
reported in 58% of the studies (Sandstrom 1996). These observations provide evidence 
for species differences in susceptibility to BKME, but also show the inherent difficulty 
when trying to compare biological responses in fish populations inhabiting highly 
different environments and exposed to complex mixtures likely to vary in chemical 
composition. In this respect, except for a decline in GSIs during the first year of study, 
the results from our field study suggest that decreased hormone concentrations in 
response to paper mill effluent exposure may not always be associated with obvious 
reproductive impairment, such as reduction in gonad weight and fecundity. Although this 
study was not designed to evaluate potential population-level effects, preliminary analysis 
would indicate no effects on age distributions and growth between exposed and reference 
populations of largemouth bass. The absence of organism-level responses is probably not 
related to a lack of sensitivity, since laboratory in vivo experiments on the impacts of 
BKME on the reproductive performance of largemouth have shown effects on gonad 






67 

weights and other measures of reproductive success (see Chapters 3 and 5). It seems 
more likely to suspect an insufficient exposure to BKME in the populations of 
largemouth bass sampled closest to the mill outfall (Rice Creek and Palatka). Although 
effluent concentrations are high in Rice Creek, the scarcity of bass in this stream would 
indicate absence of adequate prey and/or nesting substrate, thus making this area 
unsuitable for long-term residency. Fish from mainstream Palatka, on the other hand, are 
being exposed to a highly diluted effluent (less than 10% v/v) because of the high water 
flow present in the St. Johns River. 

In summary, although lower and potentially sensitive levels of biological 
organization (biochemical and physiological) were altered in largemouth bass from 
contaminated streams, these changes were not necessarily related to impacts at higher and 
less sensitive levels of organization (organ, organism, and population). It is clear then 
that additional studies are needed to further evaluate the possible impact of such 
endocrine changes in populations of Florida largemouth bass. Finally, future field study 
designs should incorporate the capability for testing relationships between chemical 
exposure and biological responses and should be accompanied by controlled laboratory 
studies that explore dose-response relationships to better interpret the data generated. 






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Figure 2.2. Mean ± SEM testosterone concentrations in largemouth bass sampled during 
pre-spawning (September 1996) and spawning (February 1997) seasons. Numbers inside 
histograms indicate sample sizes (n). There were no differences in relation to reference 
site (Welaka) (ANCOVA). 



78 



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Figure 2.3. Mean ± SEM 1 1-ketotestosterone concentrations in largemouth bass sampled 
during pre-spawning (September 1996) and spawning (February 1997) seasons. Numbers 
inside histograms indicate sample sizes (n). Asterisks indicate differences in relation to 
reference site (Welaka) (ANCOVA, Dunnett's multiple comparison test; a = 0.05). 



79 



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Figure 2.4. Mean ± SEM 176-estradiol concentrations in largemouth bass sampled 
during pre-spawning (September 1996) and spawning (February 1997) seasons. Numbers 
inside histograms indicate sample sizes (n). Asterisks indicate differences in relation to 
reference site (Welaka) (ANCOVA, Dunnett's multiple comparison test; a = 0.05). 



80 



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_H 



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1 



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#** ^ o^ v<^ 



Figure 2.5. Mean ± SEM ratio of 178-estradiol to 1 1-ketotestosterone (E/l 1-KT) in 
largemouth bass sampled during pre-spawning (September 1996) and spawning (February 
1997) seasons. Numbers inside histograms indicate sample sizes («). Asterisks indicate 
differences in relation to reference site (Welaka) (ANCOVA, Dunnett's multiple 
comparison test; a = 0.05). 






81 



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a 



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6 
5 
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3 

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0.020 



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Pre-Spawning 



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8 



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<&*' v#f c° % o 



V 



Figure 2.6. Mean ± SEM vitellogenin concentrations in largemouth bass sampled during 
pre-spawning (September 1996) and spawning (February 1997) seasons. Numbers inside 
histograms indicate sample sizes (n). Asterisks indicate differences in relation to 
reference site (Welaka) (ANCOVA, Dunnett's multiple comparison test; a = 0.05). 






82 



O 



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c 

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| 12 

fl 10 



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Mainstream 



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I 



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I 



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10 



Females 



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17 



20 



Non-detectable in 

males from exposed site 

( < 0.001 mg/mL) 



I 



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11 



18 



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6 9 



c« 



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f&* 



Figure 2.7. Mean ± SEM EROD and vitellogenin concentrations in largemouth bass 
sampled along the St. Johns River during the spawning season (March) of 1998. Fish 
were collected from tributaries or mainstream sites. Numbers inside histograms indicate 
sample sizes (n). Asterisks indicate differences of exposed tributary (Rice Creek) and 
mainstream (Palatka) sites in relation to reference streams (Cedar and Etonia Creeks for 
tributaries or Welaka and Dunn's Creek for mainstream sites) (ANCOVA, Dunnett's 
multiple comparison test; a = 0.05). 



83 



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Tributaries 



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Mainstream 



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18 



20 



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18 



X 



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X 



^ e * c V*° 



*F 



Figure 2.8. Mean ± SEM 1 1-ketotestosterone and 17B-estradiol concentrations in 
largemouth bass sampled along the St. Johns River during the spawning season (March) 
of 1998. Fish were collected from tributaries or mainstream sites. Numbers inside 
histograms indicate sample sizes (n). Asterisks indicate differences of exposed tributary 
(Rice Creek) and mainstream (Palatka) sites in relation to reference streams (Cedar and 
Etonia Creeks for tributaries or Welaka and Dunn's Creek for mainstream sites) 
(ANCOVA, Dunnett's multiple comparison test; a = 0.05). 



84 



Tributaries 



Mainstream 



3 £ 

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03 0) 

«i o 

S " 

Cm w 

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1 a 

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^ %^r ^^ &sp 



Figure 2.9. Mean ± SEM ratio of 176-estradiol to 1 1-ketotestosterone (E/l 1-KT) in 
largemouth bass sampled along the St. Johns River during the spawning season (March) 
of 1998. Fish were collected from tributaries or mainstream sites. Numbers inside 
histograms indicate sample sizes (n). Asterisks indicate differences of exposed tributary 
(Rice Creek) and mainstream (Palatka) sites in relation to reference streams (Cedar and 
Etonia Creeks for tributaries or Welaka and Dunn's Creek for mainstream sites) 
(ANCOVA, Dunnett's multiple comparison test; a = 0.05). 






85 



Tributaries 



Mainstream 





35000 - 




30000 - 


id 


25000 - 


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3 


20000 




15000 




10000 




5000 

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i 1.2 

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ec 0.6 

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re* 



^ ^° 



Figure 2.10. Mean ± SEM fecundity and egg size in female largemouth bass sampled 
along the St. Johns River during the spawning season (March) of 1998. Fish were 
collected from tributaries or mainstream sites. Numbers inside histograms indicate 
sample sizes (n). Asterisks indicate differences of exposed tributary (Rice Creek) and 
mainstream (Palatka) sites in relation to reference streams (Cedar and Etonia Creeks for 
tributaries or Welaka and Dunn's Creek for mainstream sites) (ANCOVA, Dunnett's 
multiple comparison test; a = 0.05). 



86 



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Tributary High 

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Mainstream Low/Mod 

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Mainstream High 
Oogenesis 



Males 



18 






LL y A 



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. 



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X 2 = 0.04,p = N.S. 

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Spermatogenesis 

Y//A Tributary High 
Spermatogenesis 

RXXXa Mainstream Low/Mod 
Spermatogenesis 

[ - - -| Mainstream High 
Spermatogenesis 






<$ 



£ 



Figure 2.11. Differences on the frequency of ovarian and testes development (Chi-square 
Test) in exposed largemouth bass in relation to reference. Fish were sampled along the 
St. Johns River (tributaries and mainstream sites) during the spawning season (March) of 
1998. Numbers on top of histograms indicate sample sizes (n). 



87 



Si 

0> 



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o 



20 

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Mainstream 





























20 






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Figure 2.12. Number of atretic follicles in hitological sections of ovaries from exposed 
(tributary Rice Creek and mainstream Palatka) largemouth bass in relation to reference 
(tributaries Cedar and Etonia Creeks, and mainstream Dunn's Creek and Welaka). 
Numbers inside of histograms indicates sample sizes (n). There were no differences in 
this parameter between exposed and reference sites (ANCOVA, p > 0.05). 



88 



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8 

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mS 0.04 - 

w 0.00 - 



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Y = 10.24 - 9.95(X), r 2 = 0.40, n = 13, p = 0.02 




Y = 10.49 - 5.23(X), r 2 = 0.35, n = 13, p = 0.03 



I Y = 10.50 - 1.71(X), r 2 = 0.52, n = 13, p = 0.005 



# Y = 9.88 - 0.0003(X), r 2 = 0.50, n = 13, p = 0.006 



Y = 11.44 - 77(X), r = 0.53, n = 13, p = 0.005 



5 10 15 20 

EROD (pmol resorufin/mg/min) 



25 



Figure 2.13. Regression analyses between several reproductive parameters and EROD 
activity in female largemouth bass from exposed Rice Creek and Palatka sites sampled 
during the spawning season 1998. 



89 



Reference Sites O Exposed Sites 



Females 



Males 



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Y = -8.22 + 0.04(X), r 2 = 0.67, ■ = 92, p = 0.0001 




Y =-1.9 + O.Ol(X), r 2 = 0.77, n =60, p = 0.0001 




1 1 [ 

1000 2000 3000 



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Body Weight (g) 



Body Weight (g) 



Figure 2.14. Regression lines between body length, and gonad and liver weights versus 
body weight, by sex. For gonad weight, data from spawning season 1997 was combined 
with that from spawning season 1998. For 1998, mainstream and tributary sites were 
combined. Liver weights were measured only during 1998. • = reference sites (Cedar 
and Etonia Creeks, Welaka, and Dunn's Creek); o = exposed sites (Rice Creek, Palatka, 
Green Cove, and Julington Creek). 



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a 




3 




z 


8 



4 - 



Spawning Females 



Exposed 
Reference 






Spawning Males 















i 



n 



• 






j 



i 



i 






i 



i 



i 



i 



i 



l i i i i i i 

29 34 39 44 49 54 



59 



Total Length (cm) 



Figure 2.15. Length-frequency distributions of largemouth bass, by sex. Data from 
spawning season 1997 was combined with that from spawning season 1998. For 1998, 
mainstream and tributary sites were combined. Reference sites (Cedar and Etonia Creeks, 
Welaka, and Dunn's Creek); exposed sites (Rice Creek, Palatka, Green Cove, and 
Julington Creek). 



CHAPTER 3 

IN VIVO ASSESSMENT ON THE REPRODUCTIVE EFFECTS OF PAPER MILL 

EFFLUENTS ON LARGEMOUTH BASS 



Introduction 

Results from field studies have indicated altered reproductive biomarkers for 
largemouth bass (Micropterus salmoides) sampled downstream from a paper mill in 
Florida. Fish inhabiting streams with effluent discharges had lower circulating levels of 
sex steroids (11-ketotestosterone and 17P-estradiol) and showed increased mixed- 
function oxygenase (MFO) activity. These biochemical changes, however, were not 
necessarily related to impacts at higher and less sensitive levels of organization (organ, 
organism, and population). Additional evidence showing possible endocrine alterations 
as a result of exposure to this effluent comes from work by Bortone and Cody (1999). 
These authors reported masculinization (evidenced by gonopodial development) of 
females from three poeciliid species inhabiting Rice Creek, the stream receiving the direct 
discharge from the mill. 

It has become apparent from several studies that both field and laboratory 
approaches are essential for data interpretation. Although field studies are important and 
necessary because they provide ecological relevance, they are subject to many limitations. 
For example, there is great uncertainty about exposures (doses, lengths, and routes) of 
free-ranging fish, which can seriously hinder data interpretation. In addition, the effects 
of environmental factors other than the degree of contamination on the parameters being 






91 






92 

measured are difficult to account for. Laboratory studies, on the other hand, provide the 
settings to adequately control exposure and the effects of confounding variables. 

The primary objective of the present investigation was to assess under controlled 
conditions, the effects of different concentrations of paper mill effluent being discharged 
by a plant in Palatka, on several reproductive endpoints in adult largemouth bass. 
Parameters measured in this study included body weights, lengths, and condition factor, 
organosomatic indexes, circulating concentrations of sex steroids (1 1-ketotestosterone 
and 17P-estradiol) and vitellogenin, histological development of gonads, and fecundity 
and egg sizes in females. 

Materials and Methods 

Animals and Holding Facility 

Reproductively active largemouth bass (of over 1.5yrs of age) were purchased 
from a local fish hatchery (Richloam, Terry Town, Florida) in early February 1998. Fish 
were first transported to the USGS Florida Caribbean Science Center, Gainesville, 
Florida, were they were held in fiberglass tanks for approximately one month. On March 
4, the first group of fish (56-Day exposure) was moved to Georgia-Pacific's facility in 
Palatka, and 28 days later, the remaining bass were moved for the start of the second 
exposure experiment (28-Day). In Palatka, fish were held outdoors in 10-1,500L round, 
plastic, flow-through design tanks (see Figure 3.1 for a diagram of the tank system used). 
Two additional 1,500L tanks were used to create a head pressure for each of the two 
water type treatments (well water and effluent). Head tanks were held on a 2.5m tower. 
Water used for the control tanks and for the effluent dilution was obtained from a well 



93 



located in close proximity to the tank system. Well water was pumped into a 27J50L 
pool, from were it moved into the head tank. A single, high volume, low-pressure air 
pump was used to aerate all tanks. In-line digital flow meters (ECOSOL®, Ontario, 
Canada) were set in each tank to control well and effluent inputs and enable appropriate 
effluent concentrations. Fish were fed once a week with commercial fish pellets 
("Floating Fish Nuggets", Zeigler, Gardners, PA). 
Effluent Characteristics 

Some of the chemical characteristics of the pulp bleached kraft mill effluent 
(BKME) used in this study are summarized in Table 3.1 (see National Council of the 
Paper Industry for Air and Stream Improvement, 1986 for a detailed description of the 
methodology used for the analysis of resin acids, chlorinated phenolics and phytosterols). 
The effluent tested in this study comes from a paper mill that has two bleached (40% 
product) and one unbleached line (60% product), which together release an estimated 36 
million gallons of effluent/day. The bleaching sequences for the bleach line are CEHD 
and Cc>odioEopHDp (see Chapter 2 for a description of abbreviations). The bleaching 
lines manufacture paper towels and tissue paper, whereas the unbleached line produces 
mainly kraft bag and linerboard. The wood furnish of this mill consists typically of 50% 
softwood (slash, sand, loblolly, pine) and 50% hardwood (gums, tupelo, magnolia, water 
oaks, and hickory). At the time of this study, effluents received secondary treatment, 
which consisted of both anaerobic followed by aerobic biological degradation after a 
retention period of 40 days. 



94 

Exposure Conditions 

For both lengths of exposure (28 and 56 days), approximately 40 fish were 
randomly assigned to one of five treatments: controls (were exposed to well water) and 
10, 20, 40, and 80% paper mill effluent exposures. These concentrations were chosen so 
as to cover effluent concentrations that would be in the range likely to be encountered by 
free-ranging largemouth inhabiting streams close to the Palatka mill. The average 
estimated paper mill effluent concentration in the sites closest to the mill range from 60% 
(Rice Creek) to less than 10% (Rice Creek Palatka, confluence of Rice Creek with the St. 
Johns River) (Georgia-Pacific Corporation, personal communication). However, water 
flow in Rice Creek is tidally influenced, so that during periods of low flow mill effluents 
can account for up to 90% of the total flow (Schell et al. 1993). 

Water quality measurements (temperature, pH, dissolved oxygen, salinity and 
conductivity) were taken every other day (between 10 and 12 AM) in all tanks using 
portable instruments (temperature and dissolved oxygen were measured using a YSI Inc., 
model 55, Yellow Springs, OH, USA; salinity and conductivity were measured using a 
YSI Inc., model 30, and pH was measured using a water-resistant microprocessor hand- 
held pH meter, Hanna Instruments, model H19025C, Bedfordshire, UK). A summary of 
water quality measurements is presented in Table 3.2. 
Reproductive Endpoints 

At the end of each experimental exposure (last week in April), fish were weighed, 
bled, euthanized, and necropsied as described in Chapter 2. All fish in the study were 
processed at the same time (in a 4-day period). Reproductive endpoints measured in both 
sexes included: gonadosomatic index (GSI), hepatosomatic index (HSI), sex hormones 



95 

(11-ketotestosterone and 1 7p-estradiol), vitellogenin, and histological evaluation of gonad 
development. In addition, mean egg size and fecundity were also determined in females. 
The techniques used for the measurement of these parameters in captive bass were the 
same as those described earlier for wild fish in Chapter 2. 
Statistical Analyses 

Pairwise comparisons within sex were conducted using a two-way analysis of 
covariance (ANCOVA) (PROC GLM, SAS Institute 1988) to test whether treatment 
effluent concentration and length of exposure caused significant differences in any of the 
parameters measured. Weight was used as a covariate in these analyses because fish 
exposed for 56 days were significantly heavier than fish exposed for 28 days (F = 62, p = 
0.001, and F = 64,p = 0.001 for females and males, respectively). Data sets that did not 
meet the criteria of normality and homogeneity of variance (PROC UNIVARIATE) were 
log or arcsin-transformed. If the ANCOVA showed a significant effluent concentration 
effect, a Dunnett's multiple comparison test was used to examine which effluent 
concentration(s) differed from the control group. The relationship between stages of 
gonadal development and of ovarian atresia was compared between treatments using a 
Kendall's Tau Test of association (PROC FREQ). In this test, a 95% confidence interval 
(CI) that does not include indicates a significant positive or negative relationship 
between treatment and stage of gonadal development and of ovarian atresia. For purposes 
of statistical comparisons, ovaries and testes were classified as either low to moderate 
(stages 1 and 2 for both sexes) or high gametogenesis (stages 3 for males, and 3 and 4 for 
females) (see Chapter 2 for a description of stages). In addition, ovaries were classified 
into an aditional three categories deoending on the number of atretic follicles present in 



96 

each histological section (low atresia = 1 to 15 atretic follicles; moderate = 16-25; and 
high = > 25). Statistical significance was assessed atp < 0.05. The Bootstrap regression 
model was used to calculate a point estimate, called the inhibition concentration (IC), of 
the effluent causing a 25% and a 50% reduction in the response means (IC25 and IC 50 ) as 
described in Norberg-King (1993). For example, an IC50 is the estimated concentration 
resulting in 50% inhibition relative to the control, or the estimated point where 50% of 
the population would be significantly affected, similar to the LC50. 

Results 
Females 

The effects of BKME exposure on several physiological parameters of female 
largemouth bass are summarized in Table 3.3. Exposure to effluents had no effects on 
body weights, lengths, condition factors, or HSIs. As already mentioned, however, 
females from the 56-Day group were significantly heavier and longer when compared to 
females from the 28-Day group. Gonadosomatic indexes were decreased in females 
exposed to 40 and 80% effluent concentrations for 28 and 56 days when compared to 
controls, and vitellogenin concentrations were lowered only in the 28-Day exposure 
group at all effluent levels (Figure 3.2). There was also a main effect of length of 
exposure on GSIs, with females from the 28-Day group having higher gonad weights 
(mean = 3.29%) in relation to females from the 56-Day group (mean = 2.81%). 
Vitellogenin concentrations were affected by length of exposure, with an opposite pattern 
in control females (higher concentrations in the 28-Day group in relation to the 56-Day 
group, 2.37 vs. 0.38 mg/mL) in relation to effluent-exposed bass (average of 0.21 and 



97 

0.94 mg/mL, in the 28 and 56-Day groups, respectively). Concentrations of sex steroids 
were also altered after exposure to BKME. Plasma levels of 1 1-ketotestosterone were 
reduced at exposures of 80% (28 days) and 40% and higher (56 days) (Figure 3.3), and 
17P-estradiol concentrations decreased after exposures to 20% effluent regardless of 
length of exposure (Figure 3.3). There was a significant effect of length of exposure on 
17P-estradiol concentrations, but only at the 20 and 40% effluent concentrations (mean = 
722 and 461 pg/mL for the 28 and 56 days, respectively). Concentrations of 1 1- 
ketotestosterone differed between lengths of exposure only in the 40% effluent group 
(524 pg/mL in the 28-Day group vs. 288 pg/mL in bass exposed for 56 days). The 
number of eggs produced by these females was highly variable across treatments, and 
even though there was a tendency for a decrease in fecundity with high effluent 
exposures, this trend was significant only for females exposed to high concentrations of 
BKME (40 and 80% for 28 days and 80% for 56 days) (Figure 3.4). Fecundity was also 
higher in control females from the 56-Day exposure group when compared to fish 
exposed for 28 days (mean = 12,828 vs. 8,296 eggs, respectively). Mean egg size was 
decreased only at high effluent exposures (80%) for both time periods (Figure 3.4). The 
stage of ovarian activity (expressed as level of oogenesis) was inversely related to effluent 
concentration for both lengths of exposure, although this pattern was most evident in the 
28-Day group (Figure 3.5). Similarly, the level of ovarian atresia increased with exposure 
to BKME regardless of length of exposure (Figure 3.6). Ovaries were also in a more 
advanced stage of development in females exposed to effluents for 28 days (53% of the 
ovaries examined were evaluated as highly oogenic, as opposed to 47% in the 56-Day 
group). 






98 

Males 

The effects of BKME exposure on several physiological parameters of male 
largemouth bass are presented in Table 3.4. Although body weights, lengths, and 
condition factors were not affected by exposure to mill effluents, HSIs were increased in 
males exposed to 20 and 80% effluent concentrations for 28 days, and to 20% and above 
for 56 days. As with females, males from the 56-Day exposure group had higher body 
weights and lengths when compared to males from the 28-Day group, and GSIs were 
lowered in males exposed to 40 and 80% BKME for 28 and 56 days when compared to 
controls (Figure 3.7). Vitellogenin concentrations in males averaged 0.07 mg/mL (all fish 
in the study), which corresponds to about 1/12 the concentration found in females (0.78 
mg/mL). In contrast to what was observed in females however, these concentrations were 
highly variable (in many treatments, concentrations fell below detection limit), and were 
not affected by effluent exposure (Figure 3.7). As with females, 1 1-ketotestostreone was 
decreased after exposures to high effluent concentrations (80% in the 28-Day and 40% or 
higher in the 56-Day exposure group), yet there was no clear dose-response relationship 
between effluent exposure and 1 7P-estradiol concentrations (Figure 3.8). There was also 
a main effect of length of exposure for both hormones. Whereas 1 7P-estradiol levels 
were higher in males exposed for 28 days in all treatment groups in relation to 56-Day 
males (mean = 387 and 304 pg/mL for the 28 and 56 days, respectively), concentrations 
of 1 1-ketotestosterone were increased only in the 40% effluent group (969 pg/mL in 
males exposed for 28 days vs. 471 pg/mL in the 56-Day group) (Figure 3.8). In contrast 
to what was observed in females, there was no clear relationship between exposure to 



99 

BKME and degree of spermatogenesis in testes (Figure 3.9). Finally, testes from males 
exposed to effluents for 56 days were in more advanced stages of spermatogenesis (57%) 
when compared to bass exposed to effluents for 28 days (43%). 
Both Sexes 

The ratios of 17P-estradiol to 1 1-ketotestosterone (E/l 1-KT) in plasma from 
female and male bass exposed to different concentrations of BKME are presented in 
Figure 3. 10. Overall, E/l 1-KT ratios in females averaged 2.0 indicating a predominance 
of 17P-estradiol to 1 1-ketotestosterone. In males on the other hand, this ratio fell to 
approximately a third of that found in females (0.6), indicating a predominance of 1 1- 
ketotestosterone to 17p-estradiol. For both lengths of exposure, there was a clear 
relationship between effluent concentration (20% and above) and a decline in E/l 1-KT 
ratios in female bass. Although there was a tendency for an increase in E/l 1-KT ratios in 
males exposed to effluents (20% and higher) for 56 days, this trend was not significant 
(Figure 3.10). 

A summary of inhibition concentrations (IC25 and IC50) is presented in Table 3.5. 
In males, Kiss's were generated only for 1 1-ketotestosterone (for both lengths of 
exposure) and GSIs (only for the 56-Day group). Since vitellogenin concentrations were 
highly variable in males, no IC's were calculated for this parameter. Females exposed to 
effluents for 28 and 56 days generated IC^'s for GSIs and 1 1-ketotestosterone that were 
much lower and comparable, respectively to values obtained from males (Table 3.5). 
Plasma concentrations of 17p-estradiol in females appeared to be the most sensitive 
parameter in the study, generating both IC 2 5's and ICso's, with values as low as 13% 
effluent. Declines in vitellogenin concentrations and fecundity also occurred at relatively 



100 

low BKME concentrations, but only in the 28-Day exposure group. For both sexes, there 
was an overall trend for a decline in IC values as length of exposure increased from 28 to 
56 days. The exceptions to this pattern were vitellogenin concentration and fecundity in 
females, which actually gave IC values that increased with length of effluent exposure. 

Discussion 

A summary of the reproductive responses observed in female and male 
largemouth bass exposed in vivo to BKME for 28 and 56 days are presented in Tables 3.6 
and 3.7, respectively. Overall, there was a dose-response relationship with increasing 
number of effects as the effluent concentration increased from 10 to 80%. In addition, 
several responses were intensified as length of exposure increased from 28 days to 56 
days. Except for a decline in vitellogenin concentrations in females exposed to 10% 
effluent for 28 days, all effects began at the 20% effluent concentration. 

There were similarities but also differences in the reproductive responses of 
female and male bass. In both sexes, exposure to paper mill effluents resulted in no 
changes in body weights, lengths, and condition factors. For both lengths of exposure, 
females and males responded to high effluent exposures (40 and 80%) with a decline in 
GSIs (overall declines of 39 and 22% for female and male bass, respectively). These 
declines were coupled with lower circulating levels of 1 1-ketotestosterone in bass 
exposed to these same effluent concentrations (average declines of 41% in females and of 
40% in males). Plasma concentrations of 17P-estradiol were affected differently in male 
and female bass after BKME exposure. In females, 17P-estradiol decreased after 
exposures of at least 20% effluent regardless of length of exposure (average decline of 



101 

49%), which resulted in a 37% overall decrease in E/ll-KT ratios. Changes in 17(3- 
estradiol in males, however, did not show a clear pattern in relation to effluent exposure, 
with a significant increase only in the 20% effluent group for both time periods (28% 
increase). In addition, vitellogenin concentrations were lowered only in females exposed 
to BKME for 28 days regardless of concentration (91% decline), and HSIs were increased 
only in males exposed to at least 20% effluent (16% increase). Finally, histological 
evaluation of gonads revealed changes only in ovaries (negative and positive relationship 
between effluent exposure and gonadal development and atresia, respectively), with no 
discernible effects observed in testes. 

In summary, results from the present study indicate that GSIs and 1 1- 
ketotestosterone are reduced after exposure to BKME in both sexes, but that other 
responses are sex-specific (i.e. declines in vitellogenin, 17p-estradiol, and gonad 
development were only seen in females, whereas increases in HSIs occurred only in 
males). These results are not surprising considering the fact that 17P-estradiol is not the 
main sex steroid in male bass and that vitellogenin concentrations are usually highly 
variable in this sex (see Chapter 2 for values on free-ranging fish from uncontaminated 
sites). Increases in liver weight in males could have been related to enhanced activity of 
xenobiotic biotransformation enzymes after exposure to whole effluents. In this respect, 
many studies on the effects of BKME, report a concomitant increase in HSI in fish with 
high MFO activity (Larsson et al. 1988, Munkittrick et al. 1992b, 1994, Bankey et al. 
1994, Huuskonen and Lindstrbm-Seppa 1995). Since liver weights are expected to 
increase during the reproductive season in this species and absolute values are higher in 
females compared to males (Adams and McLean 1985), pathological increases in HSIs in 






102 

females would have been harder to spot because of the concomitant physiological 
increases that are associated with vitellogenesis. Gonads were classified into four 
categories of oogenesis using a light microscope. These categories, however, were much 
more distinct when examining ovaries, which could explain the lack of discernible 
histological effects observed in males. For future studies then, testes might need to be 
examined using a more sensitive technique (e.g. electron microscopy). In addition, since 
gonadal recrudescence in this species begins slighter earlier in males than females 
(Timothy Gross, personal communication), it is also possible that any histological 
changes associated with BKME exposure might have gone undetected. 

There were differences in the endpoints measured in relation to length of 
exposure. Females from the 28-Day group had higher GSIs and fecundities, and ovaries 
were at a more advanced stage of oogenesis when compared to the 56-Day group. 
Although concentrations of sex steroids and vitellogenin also tended to be higher in 
females exposed to BKME for 28 days, this pattern was not seen in all treatment groups. 
Similarly to what was observed in females, males from the 28-Day group also had higher 
concentrations of sex steroids (particularly of 17P-estradiol). As already mentioned, fish 
from the 56-Day group were heavier and longer when compared to bass exposed for 28 
days. Since all the reproductive endpoints measured in this study are expected to increase 
with age, it is unlikely that the differences observed between both groups of fish were due 
to differences in size. A more plausible explanation for these differences has to do with 
the experimental design used in this study. With the objective of sacrificing all the fish at 
the same time (last week in April), the 28-Day group was moved to the tank facility in 
Palatka in the middle of the 56-Day dosing experiment. Prior to their movement to 



103 

Palatka, bass were held in larger tanks with clean water, which could have resulted in 
overall lower stress levels and thus higher reproductive performance in the 28-Day fish. 
It is important to mention, however, that despite the differences in the absolute values 
observed among the 28 and the 56-Day groups, the responses of both groups to effluent 
exposure were similar (except for a lack of decline in vitellogenin in females exposed for 
56 days). 

An important objective in this study was to explore the relationship between 
alterations at the biochemical level (plasma concentrations of sex steroids and 
vitellogenin) with changes at higher levels of biological organization (tissue, organ, and 
organism) after exposures to different concentrations of BKME. The results obtained so 
far look promising because they generally show that a decline in sex steroids after 
exposures to paper mill effluents results in: 1) lower gonad weights in both sexes; 2) 
increases in liver weights in males; and 3) decreases in fecundity, egg sizes, and gonad 
development in females. Although some of the biochemical-level responses were evident 
at effluent concentrations as low as 10-20% effluent (e.g. declines in vitellogenin and 
17p-estradiol in females), tissue and organ-level responses were only seen at effluent 
concentrations of 40% and above. The only exception to this pattern was the observed 
increase in liver weight in males, which started at BKME exposures of 20%. 

Inhibition concentrations were mainly calculated as a way to facilitate 
comparisons with other studies, but also as a predictive tool to identify sensitive and 
consistent responses. Changes in GSIs were more sensitive in females than males (23.5% 
vs. 69.7%). The most consistent response was observed with 1 1-ketotetosterone, with 
females and males responding with similar K^s's for both lengths of exposure. The 



104 

sensitivity of these parameters, however, was not very high since no ICso's were 
generated. In females, changes in 17P-estradiol were consistent across both lengths of 
exposure generating IC 50 's as low as 18.8%. Although changes in vitellogenin were the 
most sensitive (K^s's as low as 2.8%), there was a lack of consistency in this parameter 
with responses observed only in females exposed to effluents for 28 days. As already 
discussed, this differential response could have been related to the experimental design 
employed in this study, with higher vitellogenin concentrations in 28-Day females leading 
to more obvious declines in this parameter after effluent exposure. Fecundity appeared to 
follow more closely changes in vitellogenin than 17P-estradiol, with an increase in IC 
values with length of exposure. Finally, females responded with changes in egg sizes 
with a lack of both sensitivity and consistency. 

The results reported here are in agreement with several field and laboratory 
studies that have evaluated the impacts of BKME on fish reproductive physiology. The 
most thorough field studies on the reproductive effects of BKME have been conducted in 
Jackfish Bay, Lake Superior. From these studies, free-ranging fish exposed to paper mill 
effluents have shown declines in plasma sex steroids which have resulted in reductions in 
gonadal weights, reduced expression of secondary sex characteristics and increased age to 
maturation (Munkittrick et al. 1991, 1992a, McMaster et al. 1996a). Similar reproductive 
alterations have also been observed in fish populations sampled in proximity to other mill 
locations ( Adams et al. 1992a, Hodson et al. 1992, Gagnon et al. 1994a, 1995, Gibbons 
etal. 1998, Soimasuo et al. 1998). 

In the laboratory, fathead minnows (Pimephales promelas) exposed over their life 
cycle to BKME have also responded with decreased sex steroid production, delays in 






105 

sexual maturity, reduced fecundities, and altered secondary sexual characteristics 
(Robinson 1994). Using this same animal model but effluents from a different mill, 
Kovacs et al. (1995) reported absence of spawning in fish exposed to 20% BKME, and a 
significant delay in sexual maturation (of over 100 days) in fish exposed to 10% effluent, 
with an estimated threshold for spawning of 1.7% effluent. In addition, all of the fish in 
the study showed male secondary sexual characteristics. Full life cycle tests with fathead 
minnows have also been conducted using the BKME discharged by the Palatka operation. 
Similarly to what was observed with largemouth bass, fathead minnows exposed to 
effluent concentrations of 20% and higher had decreased sex steroids, gonad weights, and 
fecundities when compared to controls (Dennis Borton, NCASI, personal 
communication). In another laboratory study using whitefish (Coregonus lavaretus), 
exposures to 3.5% elemental chlorine free BKME has also resulted in sex steroid declines 
(40 and 37% decline in testosterone and 17p-estradiol) (Soimasuo et al. 1998). 

Results from studies on white sucker from Jackfish Bay indicate that several sites 
within the pituitary-gonadal-axis are affected after exposure to BKME. Fish from 
exposed sites had significantly lower plasma levels of gonadotropin (GtH-II) and showed 
depressed responsiveness of sex steroids and 17,206-dihydroxy-4-pregnen-3-one (17,206- 
P, a maturation-inducing steroid) after injections with gonadotropin releasing hormone 
(GnRH) (Van Der Kraak et al. 1992). BKME-exposed fish also had lower circulating 
levels of testosterone glucoronide, which would be suggestive of altered peripheral 
steroid metabolism. Similarly to what was observed under in vivo conditions, in vitro 
incubations of ovarian follicles collected from BKME-exposed females have also shown 
reduced production of testosterone, 17P-estradiol, and 17,20B-P 2 under basal and human 






106 

chorionic gonadotropin stimulated conditions (Van Der Kraak et al. 1992, McMaster et 
al. 1995). The similarities between both types of studies would suggest that reductions in 
plasma steroid levels in BKME-exposed fish from Jackfish Bay are mainly due to 
alterations in ovarian steroid production. At this point, these multiple endocrine effects 
are difficult to classify as mainly estrogenic or androgenic. This is not surprising 
considering the fact that BKME are complex mixtures capable of containing chemicals 
with simultaneous antiestrogenic, estrogenic, and even androgenic properties. 

Although there is extensive literature on the physiological effects of BKME on 
fish, very little is known about the chemical compound(s) that could be held responsible 
for such changes. Compounds such as dioxins and furans were the first to blame, because 
of their persistence, bioaccumulative properties, and their known deleterious reproductive 
effects (Safe 1999). Recent evidence, however, suggests that the chemical(s) in pulp mill 
effluents responsible for reproductive alterations are relatively short-lived and readily 
metabolized by fish. For example, mixed-function oxygenase induction and endocrine 
alterations have also been reported downstream from mills that do not use chlorine 
bleaching (McMaster et al. 1996b), and these parameters have rapidly returned to normal 
after cessation of exposure (Munkittrick et al. 1992a). Non-persistent compounds 
capable of altering the endocrine system of fish include natural wood components such as 
fatty acids (Mercure and Van Der Kraak 1995), resin acids, and plant sterols (MacLatchy 
and Van der Kraak 1995, MacLatchy et al. 1997, Tremblay and Van Der Kraak 1998, 
1999, Lehtinen et al. 1999). In this respect, chemical analyses of the effluent tested in 
this study show relatively high concentrations of natural wood components (such as plant 
sterols and resin acids, see Table 3.1), which could have been responsible for some or all 



107 

the reproductive responses observed in this study. Chlorinated compounds are also a 
concern because this mill still uses elemental chlorine in one of its bleaching lines. 
However, the concentrations of these persistent chemicals are presently low and expected 
to decrease even more after implementation of full chlorine dioxide substitution in the 
next year. 

The reproductive effects observed in largemouth bass in the present study (i.e. 
declines in sex steroids, vitellogenin, gonad weights, fecundities, and egg sizes) are 
suggestive of exposure to compound(s) capable of causing an overall inhibition of 
reproductive functions. Preliminary differential display studies using livers from bass 
exposed to this same effluent indicate changes that are different to the ones induced after 
injections with 17p-estradiol (Denslow 2000). The absence of vitellogenin induction in 
male bass exposed to this effluent is also indicative of exposure to chemical(s) that lack a 
strong estrogenic potential. As already mentioned, paper mill effluents contain a broad 
range of compounds (sterols, lignans, and resin acids) that are known to have weak 
estrogenic activity (Van Der Kraak et al. 1998). Of these, the effects of plant sterols and 
specifically of pVsitosterol on fish reproduction, have been one of the best characterized 
so far. In goldfish (Carassius auratus), injection of (3-sitosterol caused reductions in 
testosterone and 1 1-ketotestosterone in males and declines in testosterone and 17(3- 
estradiol in females (MacLatchy et al. 1997). Exposure of maturing lake trout (Salmo 
trutta lacustris) to phytosterols for 4.5 months prior to spawning resulted in increased 
dose-dependent egg mortality, smaller egg sizes and lower weight of yolk-sac larvae 
(Lehtinen et al. 1999). Antiestrogenic activity of pulp and paper mill black liquor has 
also been detected using mammalian in vitro recombinant receptor/reporter bioassays 



108 

(Zacharewski et al. 1995). There is also evidence suggesting that compounds present in 
paper mill effluents are capable of mediating endocrine responses through receptors other 
than the estrogen receptor. Female mosquitofish, Gambusia affinis, inhabiting a stream 
receiving paper mill effluents in Florida were strongly masculinized showing both 
physical secondary sexual characteristics (fully developed gonopodium) and reproductive 
behavior of males (Howell et al. 1980). More recently, masculinization of female fish 
has been identified from an additional two species (least killifish, Heterandria formosa 
and sailfin molly, Poecilia latipinna) collected from Rice Creek, the stream receiving the 
effluents discharged by the Palatka mill (Bortone and Cody 1999). Masculinization of 
female fish has been attributed to the action of androgenic hormones that result from the 
biotransformation of plant sterols (and also cholesterol and stigmasterol) by bacteria such 
as Mycobacterium (Howell and Denton 1989). The concentration of (3-sitosterol in the 
effluent under study (average of 292 ug/L, range 200 - 549 ug/L) falls within the range of 
concentrations known to affect fish reproduction. It is clear that additional studies are 
required for a better understanding of the role of plant sterols on the reproductive 
physiology of largemouth bass. 

Increases in effluent exposures in this study were associated with declines in 
dissolved oxygen and increases in salinity (see Table 3.2). Declines in dissolved oxygen 
are probably of little consequence since the lowest values observed (mean of 7.0 mg/mL 
in the 40 and 80% effluent tanks) were still well within the range tolerable for this 
species. Of more concern were the increases in salinity, which could have resulted in an 
additional source of stress for the fish (Mazik et al. 1991, Barton and Zitzow 1995). 
Stress, as mediated by the adrenal gland, has well-known effects on the reproductive 



109 

system, and might have intensified the decline in levels of reproductive hormones in fish 
exposed to high effluent concentrations (Pankhurst and Van Der Kraak 1997). Effects of 
BKME exposure on general health parameters of largemouth bass will be discussed in 
detail in Chapter 4. 

In conclusion, this work constitutes the first thorough characterization of the 
biological and physiological effects associated with exposure to paper mill effluents being 
discharged by the Palatka Operation. Largemouth bass exposed to this BKME respond 
with changes at the biochemical level (decline in sex steroids in both sexes and of 
vitellogenin in females) that are usually translated into tissue/organ-level responses 
(declines in GSIs in both sexes, and in fecundities, egg sizes, and ovarian development in 
females). The majority of these responses were observed after exposures to 20% and 
greater BKME concentrations. This threshold concentration falls within the 60% average 
yearly concentration of effluent that exists in the stream near the point of discharge, but is 
above the <10% effluent concentration present in the St. Johns River. The chemical(s) 
responsible for such changes as well as their mode(s) of action remain unknown at this 
time. 



110 



Table 3.1. Some chemical characteristics of the effluent under study. 



Chemical 


n 


Mean 


Max 


Min 


SD 


AOX (mg/L) a 


2 


7.79 


7.96 


7.61 


0.25 


TOC (mg/L) b 


3 


116 


125 


103 


11.5 


Resin Acids (ug/L) 


6 










Dehydroabietic Acid 




1,030 


2,210 


408 


702 


Abietic Acid 




1,072 


1,940 


387 


630 


Isopimaric Acid 




873 


1,490 


378 


432 


Dichlorodehydroabietic Acid 




50.1 


69.4 


41.9 


10.1 


14-Chlorodehydroabietic Acid 




35.2 


44.5 


24.3 


7.6 


Sterols (ug/L) 


7 










8-Sitosterol 




292 


549 


200 


118 


Stigmasterol 




15.6 


18.2 


12.7 


1.79 


Campesterol 




39.2 


70.5 


25.4 


14.5 


Stigmastanol 




35.6 


63.1 


25.3 


12.6 


Chlorinated Compounds (ug/L) 


11 










Phenol 




<2.0 C 








Guaiacol 




<2.0 








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2.7 


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2.7 e 


4.7 


1.5 


0.9 


3,4,5-Trichlorocatechol 




8.7 e 


13.9 


5.8 


2.5 



a = Adsorbable organic halogen. 



b 



= Total organic content. 

= All 1 1 samples were under the detection limit (2.0 ug/L). 
= Eight samples were under the detection limit (2.0 ug/L). 
= One sample was under the detection limit (2.0 ug/L) 



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119 



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Figure 3.2. Mean ± SEM gonadosomatic index (GSI) and vitellogenin concentrations in 
female largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes («). 
Asterisks indicate differences in relation to controls (ANCOVA, Dunnett's multiple 
comparison test; a = 0.05). 






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Figure 3.3. Mean ± SEM 11-ketotestosterone and 17p-estradiol concentrations in female 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes (n). 
Asterisks indicate differences in relation to controls (ANCOVA, Dunnett's multiple 
comparison test; a = 0.05). 



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Figure 3.4. Mean ± SEM fecundity and egg size in female largemouth bass exposed to 
different concentrations of paper mill effluent (0, 10, 20, 40, and 80%) for 28 or 56 days. 
Numbers inside histograms indicate sample sizes (n). Asterisks indicate differences in 
relation to controls (ANCOVA, Dunnett's multiple comparison test; a = 0.05). 



122 



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Figure 3.5. Differences on the frequency of ovarian development (Kendall's Tau Test) in 
female largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not include 
indicate a significant positive or negative association between treatment and stage of 
ovarian development (a = 0.05). Ovaries were classified into 2 main categories 
depending on degree of oogenesis. Numbers on top of bars indicate sample sizes (n). 



123 






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Figure 3.6. Differences on the frequency of atresia (Kendall's Tau Test) in ovaries from 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not include 
indicate a significant positive or negative association between treatment and degree of 
ovarian atresia (a = 0.05). Ovaries were classified into 3 categories depending on degree 
of atresia. Numbers on top of bars indicate sample sizes (n). 



124 



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Figure 3.7. Mean ± SEM gonadosomatic index (GSI) and vitellogenin concentrations in 
male largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes (n). 
Asterisks indicate differences in relation to controls (ANCOVA, Dunnett's multiple 
comparison test; a = 0.05). 



125 



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Figure 3.8. Mean ± SEM 11-ketotestosterone and 1 7p-estradiol concentrations in male 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes («). 
Asterisks indicate differences in relation to controls (ANCOVA, Dunnett's multiple 
comparison test; a = 0.05). 



126 



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Figure 3.9. Differences on the frequency of testes development (Kendall's Tau Test) in 
male largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not include 
indicate a significant positive or negative association between treatment and stage of 
testicular development (a ■ 0.05). Testes were classified into 2 main categories 
depending on degree of spermatogenesis. Numbers on top of bars indicate sample sizes 



127 



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Figure 3.10. Mean ± SEM of the ratio of 17 |3-estradiol to 11-ketotestosterone (E/ll-KT) 
in female (top) and male (bottom) largemouth bass exposed to different concentrations of 
paper mill effluent (0, 10, 20, 40, and 80%) for 28 or 56 days. Numbers inside 
histograms indicate sample sizes («). Asterisks indicate differences in relation to controls 
(ANCOVA, Dunnett's multiple comparison test; a = 0.05). 



CHAPTER 4 

IMPACT OF PAPER MILL EFFLUENTS ON LARGEMOUTH BASS HEALTH: 

FIELD AND LABORATORY STUDIES. 



Introduction 

Exposure of fish to sublethal concentrations of contaminants may impose 
considerable stress on their physiological systems, resulting in a number of manifestations 
such as reduced growth, impaired reproduction, predisposition to disease, reduced 
locomotory and predatory performance, or reduced capacity to tolerate subsequent stress 
(Adams et al. 1989). When trying to evaluate the sublethal effects of contaminants on 
fish, however, a variety of responses at several levels of biological organization are 
needed if biological and ecological meaningful results are intended. Indicators that 
reflect conditions at lower organizational levels (e.g. biochemistry) respond relatively 
rapid to stress and have high toxicological relevance; indicators that reflect conditions at 
higher organizational levels (e.g. organism, population), on the other hand, respond more 
slowly and have less toxicological but more ecological relevance (Adams et al. 1989). 

Biochemical responses offish to chemical stimuli have been studied extensively 
over the past years. The increase in monooxygenase enzyme activity (measured as 
ethoxyresorufin-O-deethylase or EROD activity) in fish livers sampled downstream of 
bleached kraft mills effluents (BKME) is one example of this (Lehtinen 1990, Martel et 
al. 1994, Huuskonen and Lindstrom-Seppa 1995). A major problem with this approach, 
however, is that the exact biological significance of these changes for the functional 

128 



129 

integrity of the organism is poorly known (Thomas 1990). In addition, factors such as 
temperature, age, sex, and nutritional status of fish can modify the activity of these 
detoxification enzymes, which could complicate the interpretation of induction responses 
in fish (Jimenez and Stegeman 1990). 

Measurements of physiological indices for assessing the effects of different 
stressors on fish are extremely valuable because they incorporate several levels of 
biological organization. Laboratory and field studies have demonstrated that exposure of 
fish to BKME can negatively affect many physiological functions. Some of these 
changes include alterations in hepatic metabolism of carbohydrates leading to disruptions 
in growth, and negative effects on hematological, immunological, and osmoregulatory 
functions (Swanson 1996). 

The main objective of this study was to evaluate, under field and laboratory 
conditions, the effects of BKME exposure on several parameters of largemouth bass 
(Micropterus salmoides). These parameters ranged from biochemical to whole organism 
levels. 

Materials and Methods 

Field Study 

Sampling sites and fish collection 

On March 1998, approximately 10 largemouth bass from each sex (total of 61 
females and 53 males) were collected by electroshocking from six sites within the St. 
Johns River (mainstream) and tributaries (small creeks) (see Figure 2.1 in Chapter 2 for a 
map showing the collection sites). Areas sampled included two tributary reference sites: 



130 

Cedar Creek located approximately 25km downstream from the mill and Etonia Creek 
which is the primary water source for the mill and is located 100-200m upstream from the 
effluent discharge, and one exposed site (Rice Creek), a small tributary stream (5 km in 
length) receiving the direct discharge from the mill. Fish were also sampled from three 
mainstream sites: reference sites Welaka and Dunn's Creek (located 40 and 18km 
upstream from effluent discharge, respectively), and exposed site Palatka (mainstream 
receiving the discharge from tributary Rice Creek). The average estimated paper mill 
effluent concentrations in the Rice Creek and Palatka sites are 60% and less than 10%, 
respectively (Georgia-Pacific Corporation, personal communication). However, water 
flow in Rice Creek is tidally influenced, so that during periods of low flow mill effluents 
can account for up to 90% of the total flow (Schell et al. 1993). Reference sites were 
matched to exposed sites in most characteristics, except presence of effluent. In order to 
minimize the variation in parameters measured in relation to timing of reproductive 
season, all fish within each site were collected within an average of four hours, and all 
sites were sampled in a 1-week period. Rice Creek was the only exception to this strict 
sampling protocol, where it was necessary to collect largemouth bass on three different 
occasions over a two-week period to achieve adequate numbers. 
Bleeding, necropsies, and age determination 

Fish were weighed using a portable digital scale to the nearest O.lg and measured 
(total length, from the tip of the mouth to the tip of the tail) to the nearest millimeter. 
Blood was collected in the field from the caudal vein using 3ml syringes and 1.5 inch, 
20G needles. Blood samples were transferred to 5ml-heparinized vacutainers and kept on 
ice until centrifugation for lOmin at 1,100 x g. Plasma was pipetted into 2mL cryotubes 



131 

and stored at -80°C until analyzed. After bleeding, fish were euthanized with a blow to 
the head, and a complete necropsy performed. Livers and spleens were excised and 
weighed to the nearest O.Olg, and hepato (HSI) and splenosomatic indices (SSI) 
calculated by dividing the weight of the organ by the weight of the fish times 100. 
Sections of spleen and liver were preserved in Notox ® for histological evaluation as 
explained below. Finally, fish were decapitated for the removal of sagittal otoliths, which 
were used for the determination of age as described in Crawford et al. (1989). 
Histopathology 

Samples of livers and spleens were collected and preserved in Notox® for 
histological evaluation. Tissues were cut transversally, embedded in paraffin, sectioned 
at 5um, mounted on glass slides, air dried and stained with Mayer's hematoxylin and 
eosin (H&E). A subsample of the sections was also stained with Perl's Prussian Blue, 
which allows for the differentiation of three pigments within melanomacrophage centers 
(MMCs): hemosiderin (ferric ion) stains bright blue, melanin appears as black to brown 
granules, and lipofucsin/ceroid pigments stain yellow brown (Blazer et al. 1987). From 
each liver and spleen section, the number of MMCs and parasites (mainly immature 
cysts) were counted. In addition, liver glycogen content and perivascular/pericanalicular 
inflammation were graded using a scale of 1 to 3 (low, moderate, and abundant). The 
presence of glycogen was verified in a subset of slides through the use of special stains 
(PAS). To reduce bias, codes for sites of collection were covered until completion of the 
histopathologic examination. 



132 

Hematological parameters and chemistry panels 

Packed cell volume (PCV) and plasma proteins were determined within an hour 
after collection of samples. For the determination of PCV, approximately 30uL of whole 
heparinized blood was collected in a 40uL microhematocrit plain capillary tube and 
centrifuged at 10,000r.p.m. for lOmin using an hematocrit centrifuge (Adams Autocrit, 
made by Clay Adams). After each PCV determination, the capillary tube was broken at 
the plasma level and two drops of plasma were applied to a portable refractometer 
(National Protometer, National Instrument Company, Baltimore, MD, USA) to determine 
the concentration of total proteins. Hemoglobin (Hb) was measured with an electronic 
hemoglobinometer (Coulter Electronics, Marietta, GA, USA). Blood was diluted (1:500) 
in isotonic diluent (Isoton HI, Coulter Electronics) and red blood cells were then lyzed 
(Zaploglobin, Coulter Electronics). The resulting solution was centrifuged at 
10,000r.p.m. for 3min to separate red cell nuclei, and the supernatant was poured into the 
hemoglobinometer. Total red blood cell (RBC) count was determined using an electronic 
particle counter (Coulter Counter model Z131, Coulter Electronics) after dilution of 
samples with isotonic diluent (1:50,000 dilution). Red blood cell size distribution range 
was selected using an electronic channelyzer (Coulter Channelyzer, Coulter Electronics). 
Osmolality was determined using a vapor pressure osmometer (Wescore, Model 5500, 
Logan, UT, USA). Two plasma samples of 5uL each were used in this technique. A 290 
mosM/L standard was run after every two fish plasma samples, and corrections to the 
concentrations obtained were made accordingly if deviations from the expected 290 value 
occurred. Chemistry panels included the determination of 12 parameters from 
approximately 350uL offish plasma: glucose; proteins (albumin and globulin); 



133 

electrolytes (sodium, chloride, potassium, calcium, phosphorous); total bilirrubin; 
creatinine; uric acid; and blood urea nitrogen. Chemistry panels were run on a Ciba 
Corning Clinical Chemistry Analyzer (Model 664 for electrolytes and Model 550 Express 
for the remaining parameters, Norwood, MA, USA). The determination of plasma 
electrolytes was given second priority when there was not enough plasma to run all 
parameters. All parameters were measured in 10 bass (5 males and 5 females) from each 
site. 
Liver enzymes 

Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline 
phosphatase (AKP) were analyzed as part of the chemistry panel and were determined 
from 10 bass/site. Liver EROD activity was determined in all fish in the study as 
described in Chapter 2. Gluathione-S-transferase (GST) activities and reduced 
glutathione (GSH) concentrations were determined using the methodology developed by 
Gallagher et al. (2000). Briefly, hepatic microsomal fractions (S-9 fractions) were 
prepared from snap frozen samples (see Chapter 2 for a detailed description of 
microsomal preparations) and stored at -80°C until analyzed. S-9 proteins were assayed 
by the BioRad protein assay kit (Richmond, CA, USA) using bovine serum albumin as a 
standard. Liver samples were kept ice-cold (4°C) throughout. Microsomal GST hepatic 
activity toward l-choloro-2, 4-dinitrobenzene (CDNB) was measured in triplicate at 30°C 
using a 96-well fluorescent microplate reader at an excitation wavelength of 544 nm and 
at an emission of 590 nm. Total hepatic GSH concentrations were determined on 
acidified, deproteinized supematants using an enzymatic recycling assay adapted for a 96- 



134 

well microplate reader. To decrease variability, GSTs and GSHs were determined only in 
males (5 males/site). 
Statistical analyses 

Pairwise comparisons were conducted using a two-way analysis of covariance 
(ANCOVA) (SAS Institute 1988) to test for differences in the dependent variables 
between sites. For this analysis, type of stream (tributary or mainstream) was used as the 
second cofactor and age was used as the covariate. Data sets from females and males 
were pooled for those parameters that were not affected by sex, and were log or arcsin- 
transformed if they did not meet the criteria of normality and homogeneity of variance 
(PROC UNIVARIATE). If the ANCOVA showed a significant site effect, a Dunnett's 
multiple comparison test was used to examine which exposed site(s) differed from the 
reference. The degree of glycogen storage and of perivascular/pericanalicular 
inflammation in livers was compared between sites using a X 2 Test (PROC FREQ). 
Statistical significance was assessed atp < 0.05. 
Laboratory Study 

Animals and holding facility 

See Chapter 3 (Materials and Method section) for a detailed description of the 
tank system facility used in this study, as well as for a chemical description of the effluent 
tested. 
Exposure conditions 

For both lengths of exposure (28 and 56 days), approximately 50 fish were 
randomly assigned to one of five treatments: controls (were exposed to well water) and 



135 

10, 20, 40, and 80% paper mill effluent exposures. These concentrations were chosen so 
as to cover effluent concentrations that would be in the range likely to be encountered by 
free-ranging largemouth inhabiting streams close to the Palatka mill. At the end of each 
experimental exposure, fish were weighed, bled, euthanized, and necropsied as already 
described. 
Physiological parameters 

Parameters and the techniques for their measurement in captive bass were the 
same as those described earlier for wild fish. Histological evaluation of spleen and 
determination of hepatic EROD, GSH, and GST were not conducted in captive bass, 
however. Body weights, lengths, condition factor, and organosomatic indexes were 
determined in all fish in the study, whereas the remaining physiological endpoints were 
measured in 10 bass/treatment (5 males and 5 females). 
Statistical analyses 

Pairwise comparisons were conducted using a two-way analysis of covariance 
(ANCOVA) (SAS Institute 1988) to test whether treatment effluent concentration and 
length of exposure caused significant differences in any of the parameters measured. 
Weight was used as a covariate in these analyses because fish exposed for 56 days were 
significantly heavier than fish exposed for 28 days (F = 62, p = 0.001 , and F = 64,p = 
0.001 for females and males, respectively). Data sets from females and males were 
pooled for those parameters that were not affected by sex, and were log or arcsin- 
transformed if they did not meet the criteria of normality and homogeneity of variance 
(PROC UNIVARIATE). If the ANCOVA showed significant effluent concentration 



136 

effects, a Dunnett's multiple comparison test was used to examine which effluent 
concentration(s) differed from the control group. 

Results 

For presentation purposes, physiological parameters were grouped into two 
categories: general health (n = 18 parameters for the field study and n = 15 for the lab 
study) and liver health parameters (n = 17 field and n = 1 1 lab). Although data on body 
weights and lengths, age, hepatosomatic indexes, and EROD are presented in their 
respective tables, further discussion on the possible effects of paper mill effluent 
exposure on these variables is omitted since it was already presented in Chapters 2 and 3. 
Field Study 

Of the 18 general health parameters studied, nine were affected by site of 
collection (Table 4.1). Splenosomatic index and number of MCCs were significantly 
decreased in bass from exposed mainstream and tributary sites (Palatka and Rice Creek), 
whereas plasma concentrations of calcium and phosphorous were increased in fish from 
these sites in relation to reference streams. Fish from Rice Creek also had lower number 
of red blood cells when compared to bass from Cedar and Etonia Creeks. Bass from 
exposed tributary and mainstream sites had increased concentrations of plasma glucose 
and creatinine, and males from the Palatka mainstream site had lower concentrations of 
cholesterol in relation to males from Dunn's Creek and Welaka (Table 4.1). 

In relation to liver health endpoints, plasma concentrations of albumin and hepatic 
concentrations of GSH were increased in males from Palatka in comparison to males 
from the reference sites (Table 4.2). Bass from Rice Creek had increased concentrations 



137 

of plasma proteins and globulin when compared to bass sampled from reference streams. 
In addition, histological examination of livers revealed comparable amounts of hepatic 
glycogen and perivascular inflammation in bass from reference and exposed sites (Figures 
4.1 and 4.2). 

A summary of significant stream effects (mainstream vs. tributary) on general and 
liver health parameters is presented in Table 4.3. Overall, half of all the parameters 
measured were affected by the size of the stream. There was no clear pattern in the case 
of general health measurements, but for liver health endpoints, all except EROD were 
higher in bass from mainstream sites. 
Laboratory Study 






The effects of different concentrations of BKME on general health parameters of 
bass after 28 and 56-Day exposures are presented in Tables 4.4 and 4.5, respectively. The 
only changes observed included a decline in the number of red blood cells in bass 
exposed to 40% effluents for 28 days (Table 4.4). Similarly, exposure of bass to BKME 
resulted in few detectable effects on liver health parameters. These changes included an 
increase in the concentration of AKP in females exposed to 40% effluents for 28 and 56 
days, and an increase in the concentration of total proteins and albumin in females and 
males exposed to at least 20% effluents for 56 days (Tables 4.6 and 4.7). 

A summary of significant length of exposure effects (28 vs. 56 days) on general 
and liver health parameters is presented in Table 4.8. As already discussed in Chapter 3, 
bass from the 56-Day group were heavier and larger when compared to bass exposed to 
effluents for 28 days. All other parameters affected by length of exposure (cholesterol, 









138 

total plasma proteins, albumin, globulin, and blood urea nitrogen) were higher in bass 
exposed to effluents for 28 days (Table 4.8). 

A rather unexpected finding came from the histological analysis of livers, which 
revealed the presence of different degrees of chronic injury (Figures 4.3 for females and 
4.4 for males). Regardless of treatment, livers of captive bass showed: accumulation of 
brown pigment by hepatocytes (which would suggest oxidative damage); fatty change 
ranging from mild cytoplasmic vacuolation to complete replacement of hepatocellular 
cytoplasm; loss of normal tissue architecture and tissue degeneration with the formation 
of regenerative nodules; mild to moderate inflammation; and increase in number and/or 
size of MMCs. These changes however, did not appear to be related to paper mill 
exposure since the frequency of distribution of lesions did not differ across treatments 
(Figures 4.3 and 4.4). 

Discussion 

Although there is a relatively large amount of information on the effects of BKME 
on health parameters of fish, most of these studies are field investigations with little or no 
information obtained from controlled laboratory studies. In addition, for many 
physiological endpoints there appears to be a lack of consistency in the responses 
observed across studies. There are several possible explanations for this, the most 
relevant being differences in: susceptibility across species; age, nutritional and 
reproductive condition of the fish being studied; chemical composition of the effluents 
tested; length of exposure; and differences in water quality parameters (such as water 



139 

temperature, dissolved oxygen, etc.) across sites. All these factors make any comparisons 
with our results difficult and point to the need for more research in this area. 

In the present study, bass sampled from the site closest to the mill discharge (Rice 
Creek) had a 63% reduction in the number of red blood cells in comparison to reference 
fish. This parameter was also lower in bass from the Palatka site, but the decline was not 
high enough to be statistically significant. In addition, bass from both exposed sites 
showed a decline in the weight of the spleen, one of the main hematopoietic tissues in 
fish. From the controlled study, the number of red blood cells appeared to decrease in a 
dose-response manner, although this decline was significant only in bass exposed to 40% 
effluents for 28 days. There was also a tendency for a decline in SSIs in fish exposed to 
at least 20% effluent for 56 days, but again this change was not significant. In both 
studies, however, lower number of red blood cells and spleen weights were not associated 
with declines in hemoglobin or PCV. 

Several field and laboratory studies have reported anemia in fish due to a decline 
in the number of red blood cells and/or in hemoglobin concentrations after exposure to 
BKME (Everall et al. 1991, Swanson et al. 1992, Khan et al. 1996, Soimasuo et al. 
1998). Lehtinen et al. (1990) used several hematological parameters to compare different 
bleaching processes through the exposure of immature rainbow trout (Salmo gairdneri) to 
effluent concentrations of 400 and 2000 times dilution for 7 weeks. From this study, fish 
exposed to effluents produced by a mill using conventional bleaching were the only ones 
to develop anemia. It has been postulated that declines in the number of red blood cells 
and hemoglobin may result from increased breakdown of red blood cells (hemolysis), 
since this phenomena has been induced in vitro after exposure of red blood cells to resin 



140 

acids (Bushnell et al. 1985). Although the exact mechanism by which resin acids cause 
hemolysis is not completely clear, they appear to cause a decrease in cellular ATP and 
oxygen consumption, leading to reduced energy production and cell death. In addition, 
there is some indication of morphological alterations (increased incidence of nuclear 
abnormalities) in red blood cells after exposure of fish to paper mill effluents, which 
could also help to explain the observed decline in blood parameters (Tripathy and Das 
1995). Decreased hemoglobin concentrations due to increased hemolysis usually result in 
elevated concentrations of bilirrubin in plasma and jaundice (Nikinmaa and Oikari 1982, 
Everall et al. 1991). The lack of changes in hemoglobin and total bilirrubin 
concentrations in largemouth bass, would suggest that the decline in number of red blood 
cells observed was caused by alterations in the hematopoietic capacity of spleens. This is 
further supported by the fact that exposed fish also had lower SSIs. Another possibility is 
that the number of red blood cells was artificially decreased due to hemodilution that 
resulted from impaired osmoregulation. As will be discussed in more detail below, this is 
an unlikely possibility since electrolytes and proteins tended to increase (concentrate) 
rather than decrease in BKME-exposed bass. 

Exposure of fish to paper mill effluents, however, has not always resulted in 
declines in blood values. Servizi et al. (1992) reported no differences in hematocrit of 
Chinook salmon (Oncorhynchus tshawytscha) exposed to up to 4% biotreated BKME for 
210 days, and Soimasuo et al. (1998) found no changes in hemoglobin and PCV in 
whitefish (Coregonus lavaretus) exposed to up to 7% BKME for a month. Similarly, 
Borton et al. (1996) exposed several species of freshwater fish species, including 
largemouth bass, to up to 8% of a high chlorine dioxide substitution BKME for 263 days 



141 

and found no effects on SSIs and hematocrit. Increases in hematocrit values probably due 
to disturbances in ion regulation and/or to stress-induced polycythemias have also been 
reported in fish sampled downstream from paper mills (Oikari et al. 1985, Hodson et al. 
1992) and in fish exposed to chlorinated compounds present in BKME (Bengtsson et al. 
1988). In a field study on the effects of BKME exposure on perch (Percafluviatilis), 
although there was a decline in hemoglobin concentrations in polluted stations, this 
decline was associated with an increase in the number of red blood cells (Larsson et al. 
1988). These authors concluded that this increased erythropoiesis was likely due to an 
increased oxygen demand as a response to the high detoxification activity associated with 
exposure to these effluents. 

Bass sampled from exposed sites had increased concentrations of plasma proteins 
(total, albumin, and globulin), cholesterol, creatinine, calcium, and phosphorous. 
Similarly, bass exposed to at least 20% effluent for 56 days also had higher concentration 
of plasma proteins (total and albumin) when compared to controls, and although not 
significant these fish appeared to have increased concentrations of plasma creatinine and 
calcium. These changes are suggestive of an osmoregulatory dysfunction commonly seen 
as a result of an adaptive stress response. The hyperglycemia observed in bass sampled 
from the Rice Creek and Palatka sites, is also considered a typical stress response 
probably associated with effluent exposure and further supports this hypothesis. 

Cortisol and adrenalin/noradrenaline are important stress hormones, which affect 
osmotic and ionic homeostasis in fish. In freshwater fish, exposure to pollutants usually 
results in a stress response with loss of ions (such as NaCl) due to an inhibition of the Na, 
K ATPase enzymes present in gills, gut, and kidney (Heath 1995a). But the osmotic and 



142 

ionic changes that are associated with stress can vary significantly depending on factors 
like genotype and body sizes, and on the presence or absence of additional physiological 
disturbances (such as vigorous activity, exposure to suboptimal water quality and physical 
injury) (McDonald and Milligan 1997). These latter conditions can lead to a significant 
lactacidosis, which in addition to induce increased gill permeability to ions and water 
(mediated by adrenaline), it causes an increase in muscle intracellular osmotic pressure 
(due to accumulation of lactate), leading to a net shift of fluid from the extracellular to the 
intracellular compartment (Milligan and Wood 1986). The impact of this transcellular 
osmotic gradient on blood parameters is to cause hemoconcentration, which is manifested 
as increases in PCV, plasma proteins and osmolality, despite net electrolyte losses to the 
water (McDonald and Milligan 1997). This complex set of responses could explain the 
wide array of electrolyte changes (increases, decreases, and no effects) observed in fish 
exposed to BKME (Lindstrom-Seppa and Oikari 1989, 1990, Oikari et al. 1988, Larsson 
et al. 1988, Lehtinen et al. 1990, Everall et al. 1991, Swanson et al. 1992, Jeney et al. 
1996). It can also help to explain the absence of significant changes in plasma 
concentrations of sodium and chloride, as well as in osmolality in the present study. A 
lack of increase in PCV could have been due to the fact that exposed bass were already 
suffering from anemia due to a decline in the number of red blood cells. 

The brain also plays a fundamental role as a regulator of osmotic function in fish. 
For example, blood calcium concentrations are mainly under hormonal regulation, with 
prolactin (secreted from the anterior pituitary) being the hormone responsible for 
stimulating the uptake of this cation from the water by the gills (Flick et al. 1984). Since 
this hormone has been found to increase significantly during acid stress in tilapia 



143 

(Oreochromis mossambicus) (Wendelaar Bonga et al. 1984) it remains unknown whether 
exposure to BKME may induce similar changes in largemouth bass. 

Cytoplasmic glutathione S-transferases (GSTs) are a multi-gene family of proteins 
that participate in detoxification processes by conjugating many electrophilic compounds 
with glutathione (GSH) to produce more soluble and thus excretable products (George 
and Buchanan 1989). Studies on the detoxification capacity of effluent-exposed fish have 
reported both increases (Oikari, et al. 1988) and declines (Mather-Mihaich and Di Giulio 
1991, Bucher et al. 1993) in hepatic GSH concentrations. GST activity, on the other 
hand, has generally been found unaltered after exposure to BKME (Soimasuo et al. 
1995a, 1995b). Similarly, an absence of GST induction in bass sampled from effluent- 
exposed streams was accompanied by a 50% increase in GSH concentrations. Increases 
in GSH, however, were observed only in bass from one of the two exposed sites 
(Palatka). As already discussed (see Chapter 2), females from the other impacted site 
(Rice Creek) showed an increased EROD activity when compared to the reference. Since 
there are several GST isoforms, it remains unknown whether the activity of at least some 
of these isozymes was indeed induced but not detected because of the very general 
substrate (CDNB) used to measure enzyme activity in this study. 

Disturbances in carbohydrate metabolism have been observed in fish exposed to 
BKME. It has been postulated that these effluents are capable of causing internal hypoxia 
through gill damage (Davis 1973), which can lead to increased blood glucose levels and 
depletion of liver glycogen. Exposure of coho salmon {Oncorhynchus kisutch) to a 
concentration of effluent equivalent to 0.8 of the 96-h LC50 produced an immediate 
hyperglycemia, and after 48h of exposure liver glycogen concentrations had decreased to 






144 

almost zero (McLeay and Brown 1975). In another study, Oikari and Nakari (1982) 
exposed trout to components of paper mill effluent for 1 1 days and observed an 
exhaustion of liver glycogen reserves. Some studies, however, have failed to detect 
changes in liver glycogen and/or blood glucose concentrations in fish after exposure to 
BKME (Oikari et al. 1988, Swanson et al. 1992, Soimasuo et al. 1998). Although we did 
observe an increase in blood glucose in bass sampled from effluent-contaminated 
streams, we observed no changes in hepatic glycogen levels in these fish. It is important 
to keep in mind however, that glycogen stores in this study were evaluated through a 
system of histological grading, which could have lacked the sensitivity present in more 
conventional methods (e.g. analysis of glucose equivalents from digested liver samples). 
Exposure of fish to BKME may increase the circulating levels of corticosteroids 
leading to immunological system disruptions, such as reductions in leuccorit and in 
immunoglobulins (Jokinen et al. 1995, Soimasuo et al. 1995a, 1995b, Khan et al. 1996). 
These changes can result in an increased susceptibility to pathogens such as bacteria and 
parasites. Kennedy et al. (1995) exposed juvenile trout to sublethal concentrations of 
chlorinated resin acids for 24 hours and observed a reduced resistance to infection by 
Aeromonas salmonicida. Several studies have also reported an increase in the prevalence 
and intensity of infection with ecto and endoparasites in fish exposed to pulp and paper 
effluents (Thulin et al. 1988, Axelsson and Norrgren 1991, Khan et al. 1992, 1994b). In 
the present study, we observed no differences in the number of immature parasitic cysts 
after histological evaluation of liver and spleen sections. This methodology, however, 
might not be the most appropriate for examination of parasite loads, and it remains 



145 

unknown whether exposure of bass to BKME resulted in changes in the gastrointestinal, 
skin, and/or gill parasitic fauna. 

There are some studies on the histopathological effects of BKME exposure in 
fish. Hepatic lesions associated with BKME exposure include: biliary hyperplasia; 
carcinomas; necrosis; fibrosis; focal vacuolation; lysosomal alterations; and loss of 
cellular compartmentalization (Lehtinen 1990, Axelsson and Norrgren 1991, Bucher et al. 
1992, Khan et al. 1994a, Teh et al. 1997). In addition, both spleens and livers of BKME- 
exposed fish have been reported to contain increased numbers of MCCs, a condition 
sometimes referred to as multifocal hemosiderosis (Khan et al. 1992, 1994a). These 
macrophage aggregates have been proposed as useful indicators of contaminant exposure 
in fish, since they collect different pigments (including hemosiderin, a breakdown product 
of red blood cells, lipofucsin, and melanin) indicative of pathological processes and tissue 
destruction (Blazer et al. 1987). In goldfish (Carassius carassim) these centers have also 
been implicated in the processing and trapping of antigens (Herraez and Zapata 1986). 
The number and size of these aggregates, however, can vary in relation to fish age, 
starvation, presence of infectious diseases, and season (Blazer et al. 1987). In this 
respect, we observed a significant positive correlation between the number of MMCs in 
liver and spleen of bass and the number of parasites in these organs (data not shown). In 
addition, bass with larger spleens and livers tended to have more MMCs. Since bass 
from exposed mainstream sites had decreased spleen weights, it is not surprising that fish 
from these sites also had a decrease number of spleen MCCs. Except for the presence of 
parasites and some perivascular inflammation, no other pathological lesions were 
observed in spleens and livers of wild bass. 



146 



Of some concern were the liver lesions observed in the experimental fish. 
Different degrees of oxidative damage, fatty changes, and tissue degeneration were 
observed in fish from all treatments, including controls. Similar lesions have been 
reported in livers from fish exposed to high concentrations of copper (Bunton et al. 
1987). Sulfites are also known to cause liver damage in fish (Ortiz et al. 1993). In a 
sample of well water analyzed during the course of the experiment, copper was present at 
a concentration of 1 ug/L and sulfates were present at a concentration of 22 mg/L. At this 
point the cause of these lesions remains unclear, but considering that control fish were 
also affected, it would suggest exposure to chemicals other than paper mill effluents. In 
addition, the physiological significance of these lesions was probably very minor, since 
they were not associated with any signs of hepatotoxicity. 

In summary, the results from this study indicate a complex pattern of effects of 
BKME on both primary (Phase I and Phase H detoxification mechanisms) and secondary 
(hematological and osmoregulatory) responses in exposed largemouth bass. Responses 
were most evident in fish from the field study, with few significant trends observed in 
captive bass exposed to different concentrations of effluents. Since most of the 
significant differences were observed in bass exposed to effluents for 56 days, it remains 
unknown whether an extended length of dosing could have resulted in increased changes, 
similar to the ones observed under field conditions. Another important conclusion from 
this study is that although many of the physiological parameters measured were 
statistically different from control or reference fish, they fell within normal physiological 
ranges when compared to reports on largemouth bass and other freshwater species 
(Denyes and Joseph 1956, Wedemeyer and Yasutake 1977, Hazen et al. 1978, Burns and 



147 
Lantz 1978, Clark et al. 1979, Borton et al. 1996). This would suggest few or no 
deleterious effects to the fish. Finally, since many of the parameters measured in this 
study are likely to be affected by a suite of environmental conditions other than chemical 
exposure (e.g. water temperature, dissolved oxygen, diet, etc.) it is essential that these 
factors not be dismissed when evaluating impacts of contaminants like BKME on 
populations of wild fish. 






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Figure 4.1. Differences on the frequency distribution of the abundance of hepatic 
glycogen and perivascular inflammation (Chi-square Test) in exposed female largemouth 
bass in relation to reference. Fish were sampled along the St. Johns River (tributaries and 
mainstream Sites) during the spawning season (March) of 1998. Numbers on top of 
histograms indicate sample sizes (n). 






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bass in relation to reference. Fish were sampled along the St. Johns River (tributaries and 
mainstream Sites) during the spawning season (March) of 1998. Numbers on top of 
histograms indicate sample sizes (n). 



163 



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Figure 4.3. Differences on the frequency of hepatic lesions (Kendall's Tau Test) in 
female largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not include 
indicate a significant positive or negative association between treatment and degree of 
liver lesions (a= 0.05). Livers were classified into 5 categories depending on the severity 
of lesions: no lesions, low, moderate, high, and severe. 



164 



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Figure 4.4. Differences on the frequency of hepatic lesions (Kendall's Tau Test) in male 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not include 
indicate a significant positive or negative association between treatment and degree of 
liver lesions (a = 0.05). Livers were classified into 5 categories depending on the severity 
of lesions: no lesions, low, moderate, high, and severe. 



CHAPTER 5 
EFFECTS OF PAPER MILL EFFLUENTS ON REPRODUCTFVE SUCCESS OF 

LARGEMOUTH BASS. 



Introduction 

Preliminary results from field studies have indicated altered reproductive 
biomarkers for largemouth bass (Micropterus salmoides) sampled downstream from a 
paper mill plant in Florida. Fish inhabiting streams contaminated with effluents had 
lower circulating concentrations of sex steroids and showed increased mixed-function 
oxygenase activity. Some of these same reproductive changes have also been replicated 
after exposure of bass to different concentrations of effluents under laboratory controlled 
conditions. In general, these findings are in agreement with several Canadian and 
Scandinavian studies which have reported alterations in reproductive 
indicators/biomarkers, including reductions in gonad size, delayed sexual maturation, and 
reduced production of sex steroids in fish sampled downstream from paper mill plants 
(Sandstrom et al. 1988, Andersson et al. 1988, McMaster et al. 1991, 1996, Munkittrick 
et al. 1992a). However, there is little understanding on whether these changes may lead 
to developmental alterations and negative reproductive success in populations of free- 
ranging fish. This lack of knowledge is surprising considering the fact that developing 
fish embryos and larvae are often considered the most sensitive stages in the life cycle of 
a teleost (Weis and Weis 1989). The effects caused at these and other stages of 



165 



166 

development by contaminants may be very subtle and go unrecognized at the individual 
level, but can have detrimental effects at the population level. 

The objective of this study was to assess the potential effects of bleached kraft 
pulp mill effluent (BKME) exposure on subsequent reproductive success of largemouth 
bass. In this study, controlled exposure of bass to different concentrations of BKME was 
followed by spawning trials that measured effects of effluent exposure on: fecundity, egg 
size, egg viability, hatchability, and fry growth and survival. 

Materials and Methods 

In Vivo Experiment 

Animals and holding facility 

Reproductively active largemouth bass were purchased from a fish farm 
(American Sportfish Hatcheries, Montgomery, Alabama) in September 1998, and 
transported to the USGS Florida Caribbean Science Center, Gainesville, Florida, were 
they were held in 0.04ha fish ponds until the start of the dosing experiment. All fish were 
moved to Georgia-Pacific's Palatka facility on December 15, where they were acclimated 
for a week prior to the effluent dosing. In Palatka, fish were held outdoors in ten 1,500L 
round, plastic, flow-through design tanks (see Figure 5.1 for a diagram of the tank system 
used). Two additional 1,500L tanks were used to create a head pressure for each of the 
two water type treatments (well water and effluent). Head tanks were held on a 2.5m 
tower. Water used for the control tanks and for the effluent dilution was obtained from a 
well located in close proximity to the tank system. Well water was then pumped into a 
series of three 27,750L pools from were it moved into the head tank. This was done in 



167 

order to increase the quality of the well water. A single, high volume, low-pressure air 
pump was used to aerate all tanks. In-line digital flow meters (ECOSOL®, Ontario, 
Canada) were set in each tank to control well and effluent inputs and enable appropriate 
effluent concentrations. Fish were fed once a week with commercial fish pellets 
("Floating Fish Nuggets", Zeigler, Gardners, PA). 
Effluent characteristics 

The effluent tested in this study comes from a paper mill that has two bleached 
(40% product) and one unbleached line (60% product), which together release an 
estimated 36 million gallons of effluent/day. Some of the chemical characteristics of this 
effluent were summarized in Chapter 3 (Table 3.1). The bleaching sequences for the 
bleach lines are CEHD and C 90 di EopHDp (see Chapter 2 for description of 
abbreviations). The bleaching lines manufacture paper towels and tissue paper, whereas 
the unbleached line produces mainly kraft bag and linerboard. The wood furnish of this 
mill consists typically of 50% softwood (slash, sand, loblolly, pine) and 50% hardwood 
(gums, tupelo, magnolia, water oaks and hickory). At the time of this study, effluents 
received secondary treatment, which consisted of both anaerobic followed by aerobic 
biological degradation after a retention period of 40 days. 
Exposure conditions 

For both lengths of exposure (28 and 56 days), approximately 50 fish were 
randomly assigned to one of five treatments: controls (were exposed to well water) and 
10, 20, 40, and 80% paper mill effluent exposures. In addition, all parameters were 
evaluated in 25 bass of each sex prior to the start of the experiment (day 0). Tanks were 
checked every other day for proper operation and for the presence of dead fish. 



168 

Water quality measurements (temperature, pH, dissolved oxygen, salinity and 
conductivity) were taken every other day (between 10 and 12 AM) in all tanks using 
portable instruments (temperature and dissolved oxygen were measured using a YSI Inc., 
model 55, Yellow Springs, OH, USA; salinity and conductivity were measured using a 
YSI Inc., model 30, and pH was measured using a water-resistant microprocessor hand- 
held pH meter, Hanna Instruments, model H19025C, Bedfordshire, UK). A summary of 
water quality measurements is presented in Table 5.1. 

At the end of each experimental exposure, fish were weighed, bled, euthanized, 
and necropsied as described in Chapter 2. Fish were sacrificed in the following order: 0, 
28, and 56 days (December 10, January 19, and February 16, respectively). 
Reproductive endpoints 

Reproductive endpoints measured in both sexes included: gonadosomatic index 
(GSI), hepatosomatic index (HSI), sex hormones (11-ketotestosterone and 176-estradiol), 
vitellogenin, and histological evaluation of gonad development. The techniques used for 
the measurement of these parameters in captive bass were the same as those described 
earlier for wild fish in Chapter 2. 
Assessment of effluent exposure 

Exposure offish to paper mill effluents was evaluated through the analysis of total 
(free and conjugated) resin acid concentrations (isopimaric, dehydroabietic, and abietic 
acids). For this analysis, bile from 10 fish/treatment (28 and 56-Day exposure groups) 
was collected and pooled by sex, and concentrations determined through gas 
chromatography/mass spectrometry (GC/MS) using the method described by Quinn 
(2000). 



169 

Statistical analysis 

Pairwise comparisons within sex were conducted using a two-way analysis of 
variance (ANOVA) (PROC GLM, SAS Institute 1988) to test whether treatment effluent 
concentration and length of exposure caused significant differences in any of the 
parameters measured. Data sets that did not meet the criteria of normality and 
homogeneity of variance (PROC UNIVARIATE) were log or arcsin-transformed. If the 
ANOVA showed significant effluent concentration effects, a Dunnett's multiple 
comparison test was used to examine which effluent concentration(s) differed from the 
control group. Temporal changes on physiological and reproductive parameters (from 
days to 56) were analyzed using a 1-way ANOVA. The relationship between stages of 
gonadal development and of ovarian atresia was compared between treatments using a 
Kendall's Tau Test of association (PROC FREQ). For purposes of statistical 
comparisons, ovaries and testes were classified as either low to moderate (stages 1 and 2 
for both sexes) or high gametogenesis (stages 3 for males, and 3 and 4 for females) (see 
Chapter 2 for a description of stages). Statistical significance was assessed at/? < 0.05. 
For those parameters that were affected by treatment, the Bootstrap regression model 
program was used to determine an effluent concentration producing a 25% and a 50% 
reduction in the response means (IC25 and IC50). 
Spawning Study 

At the end of the 56-Day in vivo exposure (mid-February), 15 males and 20 
females were collected from each of the five treatment tanks, and transported to five 
0.04ha spawning ponds located at the USGS facility in Gainesville. Approximately two 
weeks prior to the movement of fish, ponds were cleaned of all vegetation, filled with 






170 

well water, and provided with 20 spawning mats or "nests". Spawning mats ("Spawntex 
Spawning Mat", Aquatic Eco-systems, Inc., Apopka, FL) measured 61 x 51cm. Mats 
were distributed uniformly within each pond at an average depth of about 1.5m, and fixed 
to the bottom using four pieces of stainless steel wire. Ponds were completely filled with 
water after movement of fish, and thereafter monitored daily for any signs of spawning 
activity. During the course of the study, fish were fed once a week using a commercial 
pellet ("Floating Fish Nuggets", Zeigler, Gardners, PA). Dissolved oxygen, temperature, 
and pH were measured daily and averaged 8.9 mg/L, 20°C, and 8.2, respectively with no 
differences across ponds. 

Immediately after spawning behavior was detected (10 days after transfer to ponds 
males exhibited territorial behavior), mats were checked every other day for the presence 
of eggs via snorkeling. Approximately half of the mats seen with eggs were collected and 
moved to the laboratory for controlled hatchability studies (see below), while the 
remaining mats were left in the ponds for future monitoring of fry survivorship. 
Indoor hatchability studies 

In the laboratory, eggs were recovered from mats after immersion in a 1.5% 
sodium sulfite (anhydrous 97%, Acros Organics, New Jersey, USA) solution for 5 min. 
Eggs were then rinsed with tap water, and counted volumetrically to estimate fecundity 
using a graduated glass cylinder (it was estimated from several trials that 500 eggs were 
equivalent to lmL). Egg diameter was determined from each batch after measuring 30 
eggs under a dissecting scope that had an ocular metric scale. After dead eggs (white 
opaque as opposed to bright yellow) and debris were removed from each egg collection, 
viable eggs were left in fish hatching jars (Midland Fish Hatching Jar, Brookfield, WI, 



171 

USA) for a total of three days. Jars received well water at a flow rate of approximately 3 
L/min. Throughout the study, dissolved oxygen, temperature, and pH averaged 7.7 mg/L, 
21°C, and 7.6 respectively. Jars were treated daily with hydrogen peroxide (500 ppm of 
35% active ingredient, static bath for 30min, Sigma Chemical, St. Louis, MO, USA) to 
prevent fungal growth. At day three, fry were collected from each jar and counted using 
an automatic fry counter (Jensorter Fry Counter, Model FC, Bend, OR, USA). The 
number of fry produced at day 3 as a percentage of viable eggs at day was used as an 
estimate of hatchability. 
Outdoor hatchability and fry production studies 

Approximately half of the mats seen with eggs were left in the ponds to hatch 
under more natural conditions. Fry were first seen schooling on top of the mats at about 7 
days of age, but were not collected until they were 9 days old. Fry were collected using 
fry nets, and since it was difficult to collect the whole school at once, nests were visited 
every other day for up to five times. The range of fry ages collected was then 9 to 19 
days. In the laboratory, smaller fry (< 6mm) were counted using an automatic fry counter, 
whereas numbers of larger fry were counted manually. In this study, fry production per 
pond is expressed per spawned female (determined as the number of spawning mats left 
with eggs and/or fry in each pond). This correction had to be done to account for the loss 
of adult fish from ponds during the study (fish were probably preyed from the ponds by 
fish-eating birds). 
Fry measurements 

Complete batches of largemouth bass fry collected from both hatching jars and 
spawning mats were saved in 10% formalin for future measurements. From all batches, 






172 

total length was measured in 30 fry/batch, while fry weights were estimated by weighing 
four groups of 25 fry after removing excess water with a paper towel. In addition, yolk 
sac measurements (length, width, and area (estimated using the ellipsoid formula (3.14 x 
yolk length x 1 10)/6) were measured in a total of 30 three-day old fry only (collected 
from hatching jars). Finally, the frequency of gross abnormalities to the head, vertebral 
column, and yolk sac were quantified by evaluating up to 500 fry/batch. 
Reproductive parameters from spawned fish 

At the end of the spawning study (March 30), adult largemouth bass (10 of each 
sex) were collected from each pond and blood collected for the determination of sex 
steroids (11-ketotestosterone and 176-estradiol). Vitellogenin concentrations were 
determined only in females. 
Statistical analysis 

Pairwise comparisons were conducted using a one-way analysis of variance 
(ANOVA) (PROC GLM,) to test whether treatment effluent concentration caused 
significant differences in any of the following parameters: fecundity, egg size, percentage 
of live eggs, hatchability, fry production, fry measurements, and fry growth. If the 
ANOVA showed significant effluent concentration effects, a Dunnett's multiple 
comparison test was used to examine which effluent concentration(s) differed from the 
control group. The frequency distributions of different abnormalities were compared 
between treatments using a X 2 Test (PROC FREQ). 



173 

Results 

In Vivo Experiment 

Assessment of effluent exposure 

In this study, the total concentrations of three resin acids (isopimaric, 
dehydroabietic, and abietic acids) were measured in bile of bass from all treatments as a 
way to evaluate exposure to different concentrations of BKME. There was a significant 
increase in the concentrations of all resin acids in bile of fish exposed to increasing 
concentrations of effluent (Figure 5.2). In fish exposed to effluents, the overall 
concentrations of isopimaric acid were the highest recorded (mean = 17.7 ug/mL), being 
approximately twice and 4.5 times the concentration of dehydroabietic (mean = 8.8 
ug/mL) and abietic acids (mean = 4.1 ug/mL), respectively. 
Reproductive effects 

28 and 56-day exposures : The effects of effluent exposure on several 
physiological parameters of female largemouth are summarized in Table 5.2. Exposure 
to effluents had no effects on body weights, lengths, condition factors, or HSIs. Females 
exposed to effluents for 56 days had significantly higher HSIs (mean = 2.0 %) when 
compared to fish exposed for 28 days (mean = 1.5 %) (Table 5.2). Gonadosomatic 
indices were lowered in females exposed to 80% effluent concentrations for 28 and 56 
days when compared to controls, and there was no effect of length of exposure on this 
parameter (mean = 2.7 and 2.6% for females exposed for 28 and 56 days, respectively) 
(Figure 5.3). Vitellogenin decreased in a dose-response manner in both experiments, with 
significant changes beginning at lower concentrations with increasing lengths of exposure 






174 

(Figure 5.3). Effects of length of exposure on vitellogenin were significant only at the 
and 10% treatment groups with higher concentrations in the 56-Day group (mean = 2.8 
mg/mL) when compared to the 28-Day fish (mean = 1.4 mg/mL). Concentrations of sex 
steroids were also altered after exposure to paper mill effluents. Plasma concentrations of 
1 1-ketotestosterone were increased at exposures of 20% and above in female bass 
exposed for 28 days, with no changes across treatments for the 56-Day group (Figure 
5.4). In contrast, plasma concentrations of 178-estradiol decreased after exposures to 
20% (56 days) and 80% effluents (28 days) (Figure 5.4). The ratio of 176-estradiol to 11- 
ketotestosterone was reduced after exposures to 20% and above in both the 28 and the 56- 
Day groups (Figure 5.5). This decline was due to an increase in 1 1-ketotestosterone and a 
decline in 176-estradiol in females exposed to effluents for 28 and 56 days, respectively. 
There was an overall increase in sex steroid concentrations from days 28 to 56 (from a 
mean of 217 to 602 pg/mL and from 522 to 968 pg/mL, for 1 1-ketotestosterone and 176- 
estradiol, respectively). The stage of ovarian development (expressed as degree of 
oogenesis) was inversely related to effluent exposure but only in the 28-Day group, with 
no significant histological changes observed in fish exposed for 56 days (Figure 5.6). The 
degree of ovarian atresia was inversely and directly related to effluent exposure in 
females exposed for 28 and 56 days, respectively (Figure 5.7). 

The effects of effluent exposure on several physiological parameters for male 
largemouth are presented in Table 5.3. Although body weights, lengths, and condition 
factors did not differ across treatments in the 28-Day group, males from one of the 
treatments (40%) in the 56-Day group tended to be slightly larger and heavier when 
compared to controls. This increase in size, however, did not result in differences in 



175 

condition factor. Similarly to what was observed in females, HSIs were not affected by 
exposure to BKME, but increased from a mean of 1.29 % in the 28-Day group to a mean 
of 1.73 % in males exposed for 56 days (Table 5.3). Gonadosomatic indices were 
lowered in males exposed to 80% effluent concentrations for 28 and 56 days when 
compared to controls (Figure 5.8), and there was an unexpected increase in this index in 
males exposed to 20% effluent for 56 days. Effects of length of exposure on GSIs were 
significant only at the 0, 10, and 20% treatment groups with a higher index for the 56-Day 
group (mean = 1.1 %) when compared to the 28-Day fish (mean = 0.8 %). Vitellogenin 
concentrations in males averaged 0.1 1 mg/mL (all fish in the study), which corresponds 
to about 1/12 the concentration found in females (1.3 mg/mL). Plasma concentrations of 
this protein increased with length of exposure from an average of 0.07 mg/mL in males 
exposed for 28 days to 0.21 mg/mL in males exposed for 56 days. In contrast to what 
was observed in females however, these concentrations were highly variable (in many 
treatments concentrations fell below detection limit), and were only affected after 
exposure to high effluent concentrations (80%) for 56 days (Figure 5.8). 1 1- 
ketotestosterone decreased in a dose-dependant manner in both experiments, with 
significant effects beginning at lower concentrations with increasing lengths of exposure 
(Figure 5.9). On the other hand, 176-estradiol tended to increase in fish exposed to 
effluents (>20%) for 56 days. In contrast to what was observed in females, these 
hormonal changes resulted in increases in E/l 1-KT ratios in males exposed to 40% 
effluent and above for both lengths of exposure (Figure 5.5). There was also a main 
effect of time of exposure for both hormones, with concentrations almost duplicating in 
males from the 56-Day group when compared to males from the 28-Day group (mean = 



176 

506 and 880 pg/mL for 1 1-ketotestosterone and mean = 306 and 572 pg/mL for 178- 
estradiol). Similarly to what was observed in females, the stage of testicular development 
(expressed as degree of spermatogenesis) was inversely related to effluent exposure but 
only in the 28-Day exposure group, with no significant histological changes observed for 
the 56-Day group (Figure 5.10). 

Post-spawning fish : Concentrations of sex steroids and vitellogenin in females 
were measured in post-spawned bass (March 30) that had been exposed to different 
concentrations of paper mill effluents for 56 days. After a depuration period of 42 days, 
concentrations of sex steroids in females did not differ across treatments (Figure 5.1 1), 
and even though vitellogenin concentrations tended to be higher in females that had been 
exposed to high BKME, this change was significant only for the 40% treatment group 
(Figure 5.12). Males however, still showed altered concentrations of sex hormones, with 
a significant decrease and increase of 1 1-ketotestosterone and 176-estradiol, respectively 
after exposures to effluent concentrations of 20% and higher (Figure 5.11). These 
hormonal changes resulted in increases in E/l 1-KT ratios as shown in Figure 5.12. 

Temporal changes : The objective of this analysis was to compare changes in 
reproductive parameters in largemouth bass throughout the reproductive season, from 
December 10 (pre-spawning, day 0) through February 16 (day 56) in relation to exposure 
to paper mill effluents. In females, GSIs increased about 33% from December to January 
(day 28), but only when exposed to concentrations of 20% effluent or less; females in the 
40 and 80% treatment groups showed no significant increases in GSIs (< 10% increase) 
in relation to day (Figure 5.13). Females sampled in February (day 56) showed a 
similar pattern, with increases in GSIs in all treatments (23% increase overall) except the 









177 

80% effluent exposure group. Vitellogenin concentrations were increased only in the 56- 
Day group that was exposed to and 10% effluent, and females exposed to higher 
effluent concentrations tended to have lower vitellogenin when compared to pre- 
spawning bass (Figure 5.13). Seasonal changes in 1 1-ketotestosterone did not appear to 
be related to effluent exposure, and were most apparent in females sampled in February 
(over 100% increase from day 0) (Figure 5.14). Although there was a tendency during 
the January (day 28) sampling for females exposed to high effluent (80%) to have 
concentrations of 176-estradiol that were lower than those observed during December, 
declines in percent change of this sex steroid were most evident in the 56-Day group after 
exposures to 40 and 80% effluent (Figure 5.14). 

In males, GSIs increased from December to January, but only in bass that had not 
been exposed to BKME (Figure 5.15). In February, this index had increased considerably 
in the 0, 10, and 20% treatment groups (an overall increase of 84%), with lower (20%) or 
no increases in the 40 and 80% exposure groups, respectively. Seasonal changes in 
vitellogenin concentrations in plasma of males are presented in Figure 5.15, and behaved 
similarly to what was described for GSIs. Concentrations of 1 1-ketotestosterone in males 
sampled in January were increased only in controls in relation to males sampled in 
December (Figure 5.16). During February, concentrations of this sex steroid increased 
regardless of treatment, although were highest in bass from the control and 10% effluent 
exposure groups. In contrast to what was observed with 1 1-ketotestosterone, 
concentrations of 176-estradiol remained more or less constant in the 28-Day group, but 
tended to increase with effluent exposure in males sampled in February (Figure 5.16). 



178 

Inhibition concentrations : A summary of inhibition concentrations (IC25 and IC50) 
for all reproductive parameters measured in this study is presented in Table 5.4. In males, 
IC25's were generated for both lengths of exposure only for 1 1-ketotestosterone and GSIs. 
Since vitellogenin concentrations were highly variable in males, no ICs were calculated 
for this parameter. Females exposed to effluents for 28 and 56 days generated K^s's on 
GSIs and 178-estradiol that were as low as 15.4 and 17.5% effluent, respectively. 
Changes in plasma concentrations of vitellogenin in females appeared to be the most 
sensitive parameter in the study, generating both Kiss's and ICso's, with values as low as 
13% effluent. For both sexes, there was an overall trend for a decline in IC values as 
length of exposure increased from 28 to 56 days. 
Spawning Study 

A summary of spawning mat activity and number of fry produced is presented in 
Table 5.5. The number of mats collected for the indoor hatchability study averaged 5 
across ponds (range 2 - 7), which ended up corresponding to about 25% of the total 
number of mats present in each pond. Originally, we intended to collect approximately 
half of the mats (i.e. 10 mats) seen with eggs for these studies, but because of water 
visibility problems the number of mats that were actually seen with eggs in each pond 
was reduced. This was particularly evident in the case of the 40% pond from which only 
four mats were seen with eggs (and two collected) at some point during the study (Table 
5.5). From the indoor hatchability study, the average number of fry produced per 
spawned female was similar across treatments and ranged from 1,978 for the 80% pond, 
to 3,881 for the 10% pond (age 3 days). Although about 75% of the mats originally 
installed in each pond remained there available for bass, less than half of them (average 



179 

of 45%) were used. From the outdoor study, the average number of fry produced per 
spawned female (age 14 days) decreased with effluent exposure from almost 4,000 fry in 
the control group, to less than 100 in the 80% group. When the results of both studies 
were combined however, the average fry produced per spawned female was significantly 
reduced only in the 40 and 80% effluent groups. There was also some adult mortality 
throughout the study, particularly of females (average loss of 8 females/pond), which was 
probably due to predation by fish-eating birds (Table 5.5). 
Indoor hatchability studies 

Fecundities, egg sizes, percentage of live eggs, and hatchabilities did not differ 
across treatments and averaged 7,104 eggs, 1.32mm, 78%, and 47%, respectively (Figure 
5.17). Fry produced from BKME-exposed bass had yolk sacs that were of similar length 
(mean = 1 1.3mm), but had widths that tended to decrease with effluent exposure (from a 
mean of 7.7mm in the control group to a mean of 7.3mm in the 80% group) (Figure 5.18). 
This slight decrease in yolk sac width, however, did not result in changes in yolk sac area 
(overall mean of 44.1mm ). Although fry measured at day 3 were of similar length across 
ponds (mean = 5.6mm), there was a tendency for a decline in body weights, but only at 
the 40 and 80% effluent treatment groups (mean =1.3 and 1.1 mg for the to 20% and 40 
and 80% groups, respectively) (Figure 5.19). In addition, the frequency of fry 
abnormalities increased from an average of 10.5% in the through 40% effluent groups, 
to almost 17% in the 80% effluent group. The distribution of fry abnormalities was 
similar in the first three treatment groups, but the abnormalities to the head tended to 
increase in the 40 and 80% treatment groups (mean of 5.3% as opposed to 0.73% in the 
control, 10, and 20% effluent groups) (Figure 5.19). 



180 

Outdoor hatchability and fry production studies 

There was a dose-response decline in fry production with exposures to increasing 
concentrations of effluent (Figure 5.20). This decline was of almost 300% for the 10, 20, 
and 40% effluent groups, and of over 4,000% for the 80% treatment group in relation to 
controls. Fry weights and lengths also decreased in a dose-dependant manner from an 
average of 3.8mg and 7.8mm in the control group to 2.7mg and 6.8mm in fry produced by 
adult bass exposed to BKME (Figure 5.21). Although fry from the 20% effluent group 
tended to be smaller when compared to the control group, this difference was not 
statistically significant. In contrast to what was observed in 3-day old fry collected from 
hatching jars, there was no association between effluent exposure and frequency of 
abnormalities in 14-day old fry collected from ponds (overall mean of 1.5% 
abnormalities, about 8 times lower than in the 3-day old fry) (Figure 5.21). Overall fry 
growth from days 9 to 19 in relation to effluent exposure is presented in Figure 5.22. 
This analysis only includes mats from which repeated measurements of fry were taken at 
intervals of about two days. There was a decline in fry growth only for the 40 and 80% 
effluent groups. 

Discussion 

In Vivo Experiment 

In this experiment, we were able to determine exposure to BKME through the 
measurement of total (free and conjugated) resin acids in bile. The concentrations of 
isopimaric and dehydroabietic acids in bile of largemouth bass increased in relation to the 
mean percentage dilution of BKME used in the different treatments. Abietic acid, on the 



181 

other hand, did not follow the same trend and increased to similar concentrations 
regardless of effluent dilution. Resin acid concentrations in bile of fish have been used as 
a biomarker of exposure to paper mill effluents, and the values observed in this study are 
comparable to those reported from whitefish (Coregonus larvaretus) and rainbow trout 
{Oncorynchus mykiss) caged at increasing distances from paper mills (Oikari and 
Kunnamo-Ojala 1987, Leppanen et al. 1998). Although fish can readily build up body 
burdens of resin acids after waterborne exposures, depuration rates are also known to 
occur fast, with half-lives of less than 4 days (Niimi and Lee 1992). Preliminary results 
from largemouth bass also show relatively rapid depuration rates, with non-detectable 
concentrations in fish from the 80% effluent group measured 42 days post-treatment (data 
not shown). These results suggest that measuring resin acids in bile of free-ranging bass 
might be a useful indicator of short-term exposure to paper mill effluents, but that 
measurements of more persistent compounds (e.g. chlorinated organics) are probably 
needed for a better assessment of chronic exposures. 

A summary of the reproductive responses observed in female and male 
largemouth bass exposed in vivo to BKME for 28 and 56 days are presented in Tables 5.6 
and 5.7, respectively. Overall, there was a dose-response relationship with increasing 
number of effects as the effluent concentration increased from 10 to 80%, with many 
responses being intensified as length of exposure increased from 28 to 56 days. Most 
effects began at the 20% effluent dilution, and sex steroid concentrations were back to 
normal in post-spawned females after a depuration period of over 40 days, but remained 
altered in males. 



182 

There were similarities but also differences in the reproductive responses of 
female and male bass exposed to effluents for 28 and 56 days. In both sexes, exposure to 
BKME resulted in no changes in body weights, lengths, and condition factors. For both 
lengths of exposure, females and males responded to high effluent exposures (80%) with 
a decline in GSIs (overall declines of 22 and 35% for female and male bass, respectively). 
In addition, histological evaluation of gonads revealed changes in both ovaries and testes 
(negative relationship between effluent exposure and gonadal development), but only in 
fish exposed for 28 days. There was also an increase in the number of atretic follicles in 
females exposed to 40 and 80% effluents for 56 days, which could be suggestive of 
toxicity. As will be discussed in more detail later, however, these pathological changes 
did not result in a decline in the number of eggs spawned. Increase in the numbers of 
atretic follicles have also been reported from ovaries of rainbow trout exposed to 
pentachlorophenol for 18 days (Nagler et al. 1986), a chemical known to be present in 
BKME, and in female redbreast sunfish (Lepomis auritus) sampled from a BKME- 
impacted river in Tennessee (Adams et al. 1992). 

Plasma concentrations of sex steroids and vitellogenin behaved differently in male 
and female bass after BKME exposure. In males, there was a dose-response decline in 
1 1-ketotestoterone starting at 20% effluent (average decline of 29%), whereas 178- 
estradiol increased in the 56-Day group (37% increase) after exposure to similar 
concentrations of effluent. Interestingly, these endocrine alterations persisted in post- 
spawned males 42-days after the end of the dosing experiment. In females, vitellogenin 
and 178-estradiol decreased in a dose-dependant manner after exposures to at least 20% 
effluent (67 and 36% declines, respectively), whereas 1 1 -ketotestosterone was increased 



183 

in the 28-Day group (38%) after exposures to similar effluent dilutions. In contrast to 
what was observed in males, sex steroid concentrations had returned to normal in post- 
spawned females after a depuration period of over a month. A discussion on similar 
reproductive alterations observed in fish exposed to BKME elsewhere, as well as on 
possible causative chemicals present in these effluents and their mode of action has 
already been presented in Chapter 3. 

The presence of endocrine alterations in male bass after cessation of exposure to 
BKME is suggestive of exposure to persistent chemicals. Elimination of important body 
burdens of lipophilic compounds through the production of eggs could also explain the 
absence of similar endocrine changes in post-spawned female bass. In this respect, there 
is evidence showing translocation of dioxins such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin 
(TCDD) from adult female fish to oocytes (Wannemacher et al. 1992, Walker et al. 1994) 
and of exposure of bass to this compound downstream from the Palatka mill (Schell et al. 
1993). The endocrine disrupting properties of TCDD and related halogenated aromatic 
hydrocarbons are well documented (Peterson et al. 1993), and as discussed below could 
explain some of the reproductive effects observed in this study. 
Spawning study 

A summary on the significant changes observed during the spawning study is 
presented in Table 5.8. The observation that the effluent being released by the Palatka 
mill was capable of causing endocrine alterations in adult largemouth bass led us to the 
implementation of spawning studies with the objective of evaluating the reproductive 
consequences of such changes. Full-life cycle tests measuring the effects of 
environmental contaminants are important because they provide ecological relevant 






184 

information that can then be applied in risk assessments models. A practical limitation of 
these tests, however, is that they usually require that fish reach sexual maturity at a young 
age (e.g. 12 or 25 weeks for the commonly used fathead minnows, Pimephales promelas, 
and zebrafish, Danio rerio, models) (Kovacs et al. 1996, Nagel and Isberner 1998). Since 
Florida largemouth bass do not reach sexual maturity until they are at least 8 to 9 months 
old, (Hardy 1978) implementation of full-life cycle tests in this species are limited. The 
methodology described in the present study offers a good alternative for evaluating the 
potential effects of BKME and other chemicals on early life stages (egg and fry) of bass. 

Our original hypothesis was that the reproductive changes observed in BKME- 
exposed adult largemouth bass were going to result in: i) delayed or absence of spawning 
and altered reproductive behaviors; ii) decreased fecundities and egg sizes; iii) decreased 
hatchabilities; and iv) decreased fry growth and survival. Surprisingly, and despite the 
observed declines in sex steroids and vitellogenin, bass from all treatments began 
spawning approximately 10 days after they were moved to clean water ponds. Males 
showed aggressive territorial behavior (i.e. biting and chasing snorklers away) regardless 
of treatment, although this was not quantified. In addition, almost two months of effluent 
exposures did no affect fecundities, egg sizes, percentage of live eggs, and hatchabilities, 
which at the end was translated into similar numbers of fry produced across treatments. 
At this stage (3 days of age) fry were also measured and examined for the presence of any 
gross abnormalities. Here it became apparent that fry produced by bass exposed to high 
BKME concentrations (40 and 80%) were suffering from the effects of such an exposure 
because they tended to be smaller and show a higher frequency of deformities. We were 
also interested in measuring some of these same parameters in older fry (average age of 



185 

14 days) hatched and grown under more semi-natural conditions (under the effects of 
different pond stressors). Results from this part of the study showed significant negative 
effects of effluent exposure on fry growth and survival. 

There are relatively few studies on the effects of BKME on egg parameters, and 
the results from these studies are conflicting. In contrast to what was observed in the 
present study, fertilities (as indicated by the percentage of spawned eggs that hatched) 
were decreased in zebrafish after exposure to chlorinated phenolics from a bleach plant 
effluent (Landner et al. 1985) and in brown trout (Salmo trutta) after exposure to BKME 
(Vuorinen and Vuorinen 1985). Hatchabilities were also reduced in pike (Esox lucius) 
after exposure of eggs to BKME concentrations as low as 0.5% (Tana and Nikunen 
1986). Similarly, many field and laboratory studies have reported declines in fecundities 
of several fish species after exposure to paper mill effluents (Landner et al. 1985, 
Munkittrick et al. 1991, Gagnon et al. 1994b, 1995, Kovacs et al. 1995). McMaster ef a/. 
(1992) on the other hand, found equal or greater fertilization rates and no effects on 
hatchabilities of eggs of white suckers after BKME exposure, despite declines in sex 
steroid concentrations, gonad and egg sizes and sperm motility in these fish. In addition, 
fecundities and/or hatchabilities were not altered after exposures to BKME in several 
other field (Karas et al. 1991, Swanson et al. 1992, Adams et al. 1992) and laboratory 
studies (Kovacs et al. 1996). Exposure of female bass to paper mill effluents in this study 
caused important declines in plasma concentrations of 1 7P-estradiol and vitellogenin, but 
these declines were not associated with reduced fecundities, egg sizes, or hatchabilities. 
A possible explanation for this lack of association could be related to the timing of 
exposure. Because vitellogenesis in Florida largemouth bass starts in September and 



186 

peaks in January (Timothy Gross and Nancy Desnlow, unpublished data), by the time our 
experiments started females had already allocated a considerable amount of vitellogenin 
in the developing oocytes. It remains unknown whether a more prolonged period of 
dosing, extended to cover most of the oocyte growth phase could have resulted in 
alterations in some or all of the egg parameters measured in this study. 

From the outdoor experiment, there was a significant decline in the average 
number of fry produced per spawned female, starting at 10% effluent exposures. 
Declines in the numbers of fry produced were probably not due to decreased fecundities 
or hatchabilities, since results from the indoor study showed no effects of effluent 
exposure on these parameters. It seems more likely to assume that declines in fry 
production resulted from increases in fry deformities and delayed growth rates. Such 
impairments may be critical for survival, since the susceptibility to several environmental 
factors (such as water quality parameters and exposure to contaminants) may be increased 
and larvae may not be able to swim for food properly. In this respect, the transition 
period between internal and external feeding during the early yolk sac stage is generally 
recognized as one of the most sensitive to the action of toxicants (Westernhagen 1988). 
Feeding by largemouth bass larvae starts at around 8 days of age (Chew 1974), which 
corresponds to the approximate age of observed decreased survival in this study. 

There is very little information on the developmental effects of BKME. In the 
laboratory, survival from larvae to adult and growth of fathead minnows were not 
affected after exposures to up to 20% effluent concentrations (Kovacs et al. 1995, 1996). 
In addition, in one of these studies effluents caused no morphological or histopathological 
abnormalities in hatched fish. Similarly to what was observed with largemouth bass, 






187 



Karas et al. (1991) reported comparable fecundities and egg mortalities in perch (Perca 
fluviatilis) from a BKME-exposed area, but fry hatched from this site were smaller and 
had an increased frequency of abnormalities which was translated into lower abundances 
of fry and young-of-the-year fish (Karas et al. 1991). Lack of food could not explain the 
increased mortalities because feeding conditions were found to be similar between 
exposed and reference sites. These authors concluded that exposure of perch to BKME 
had resulted in high mortality rates close to the time of hatching due to either chronic 
failure of parental reproductive systems and/or acute toxicity to embryos or early larvae. 
Exposure of developing bass embryos to chemicals present in the effluent tested 
could explain the increased frequency of deformities and retarded growth observed. It is 
well known that pesticides and many industrial hydrocarbons can be highly lipophilic and 
may concentrate in lipid-rich tissues such as liver and ovaries (Kime 1995, Heath 1995b). 
This may lead to the incorporation of contaminants into the developing oocyte, which in 
turn may be translated into increased frequencies of deformities and decreased growth 
rates in the offspring. We observed an increase in fry deformities at the highest 
concentration tested (80%), but only in the 3-day old group. The frequency of these 
abnormalities, however, was significantly reduced in the 14-day old fry with no trends 
across treatments. This decline is not surprising since it is likely that most of these 
morphological changes were associated with lethalities at an early age. Exposure of the 
developing embryo to toxicants can also lead to sublethal effects such as reductions in 
growth rates. Heavy metals, petroleum hydrocarbons and chlorinated hydrocarbons are 
known to cause reduced length of newly hatched larvae, phenomena that is frequently 
correlated with larger yolk-sac sizes (Westernhagen 1988). The size of the yolk sac in fry 



188 

has been used as an indicator of yolk utilization, with increased values suggestive of 
impaired development. Except for a slight decline in weight of 3-day old fry from the 40 
and 80% effluent groups, there was no other indication of impaired growth at this stage 
(fry lengths and yolk sac areas did not differ across treatments). Assuming that lengths 
and yolk sac sizes of newly hatched larvae were similar across treatments for fry hatched 
outdoors, these results would suggest that alterations in growth after effluent exposure are 
only likely to be noticeable if measured later on during development. 

Chemicals found in BKME that are capable of being translocated from the mother 
to the developing oocyte include chlorinated organics such as TCDD (Wannemacher et 
al. 1992, Walker et al. 1994), but also naturally occurring wood-derived compounds like 
phytosterols (Lehtinen et al. 1999). Similarly to what was observed in BKME-exposed 
bass, these studies have also reported declines in fry survival due to retarded growth 
coupled with increased prevalence of deformed larvae after exposure of adult fish to these 
compounds. Although no chemicals were measured in either eggs or fry in the present 
study, there is evidence showing that aqueous uptake does not play a major role in the 
bioaccumulation of dioxins and furans, with ingestion via the food chain being the 
dominant mechanism (Rogers et al. 1989, Servizi et al. 1992). It is clear that more 
studies are needed for a better understanding on the chemical(s) responsible for the 
observed alterations, as well as on their mode of action. 

Another possible factor that could have contributed to the observed changes in fry 
relates to alterations in the "quality" of the yolk that was being deposited in the 
developing oocyte during the course of the in vivo exposures. Vitellogenesis refers to the 
production of egg yolk and involves the mobilization and transport of lipids, metals, ions, 



189 

vitamins, and hormones to the fish ovary (Specker and Sullivan 1993). For example, 
essential metals such as zinc, copper, calcium, magnesium, and iron are transported to the 
egg yolk via the highly charged and abundant phosphate groups of the phosvitin region of 
the vitellogenin molecule (Richards and Steele 1987). It has also been proposed that 
vitellogenin may serve as an important carrier molecule for steroid (such as Cortisol and 
sex steroids) and thyroid hormones into the developing ovary. Although the 
physiological significance of this transfer remains largely unknown, it has been postulated 
that maternally derived steroid hormones can influence several developmental processes, 
including sexual differentiation (Schreck et al. 1991). Decline in the production of 
vitellogenin by livers of BKME-exposed females could have resulted in concomitant 
decreases in the amounts and types of essential nutrients and possibly hormones that were 
being mobilized into the developing egg, which could have negatively affected the 
normal development of fry. 

One of the main objectives of controlled laboratory studies is to estimate threshold 
concentrations capable of causing specific alterations. Once effluent concentrations in 
the receiving streams are known, the next step involves caution extrapolation of results 
obtained under controlled conditions for use in evaluations of impacts in the field. It is 
important to keep in mind, however, that these extrapolations are subject to many 
limitations, including uncertainties regarding impacts at higher levels of biological 
organization (population, community, and ecosystem). In this respect, the results from 
our spawning study suggest negative effects of BKME on fry growth and survival with a 
threshold concentration of 10%. This threshold concentration falls within the 60% 
average yearly concentration of effluent that exists in the stream near the point of 



190 

discharge (Rice Creek). These findings then would suggest probable population-level 
effects mainly in fish inhabiting this small stream. However, since the lowest effluent 
concentration tested in this study was 10%, it remains unknown if bass inhabiting areas of 
lower effluent concentration could potentially be affected by such an exposure as well 
(e.g. at the confluence of Rice Creek with the St. Johns River effluents are estimated to be 
under 10%). In addition, because many reproductive responses observed in BKME- 
exposed bass were intensified with length of dosing, exposures of over 56 days could 
result in increasingly lower threshold concentrations, leading to population-level effects 
in low-level effluent streams. 





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1 #««#**«#»■ 




Figure 5.1. Diagram of the tank system used for the in vivo exposures. Each fish tank 
has a 1,500L capacity. 



203 




0% 10% 20% 40% 80% 
Effluent Concentration 



Figure 5.2. Mean ± SEM total (free and conjugated) resin acid concentrations in bile of 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%). Each bar represents pooled samples from 5 females and 5 males from both the 
28 and the 56-Day exposures. IPA = isopimaric acid, DHAA = dehydroabietic acid, AA 
= abietic acid. Asterisks indicate differences in relation to controls (ANOVA, Dunnett's 
multiple comparison test; a = 0.05). 



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204 



56 Days 



20 



19 



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28 



* 

X 
18 




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19 



0% 10% 20% 40% 80% 



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Figure 5.3. Mean ± SEM gonadosomatic index (GSI) and vitellogenin concentrations in 
female largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes (n). 
Asterisks indicate differences in relation to controls (ANOVA, Dunnett's multiple 
comparison test; a = 0.05). 






205 



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200 


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Figure 5.4. Mean ± SEM 11-ketotestosterone and 1 7(3-estradiol concentrations in female 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes (n). 
Asterisks indicate differences in relation to controls (ANOVA, Dunnett's multiple 
comparison test; a = 0.05). 



1 1.5 

g 

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206 



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121 



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Figure 5.5. Mean ± SEM of the ratio of 1 7p-estradiol to 1 1-ketotestosterone (E/l 1-KT) 
in female (top) and male (bottom) largemouth bass exposed to different concentrations of 
paper mill effluent (0, 10, 20, 40, and 80%) for 28 or 56 days. Numbers inside 
histograms indicate sample sizes (n). Asterisks indicate differences in relation to controls 
(ANOVA, Dunnett's multiple comparison test; a = 0.05). 



207 



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19 



20 



17 



19 



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0% 10% 20% 40% 80% 

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Figure 5.6. Differences on the frequency of ovarian development (Kendall's Tau Test) in 
female largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not include 
indicate a significant positive or negative association between treatment and degree of 
ovarian development (a = 0.05). Ovaries were classified into 2 main categories 
depending on degree of oogenesis. Numbers on top of bars indicate sample sizes («). 



208 



Low 



Moderate 






100 
80 
60 
40 
20 


100 



£ 80 

I 60 

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56 Days Kendall's Tau 95% CI (0.20, 0.52) 



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15 



40 

20 






0% 10% 20% 40% 80% 
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Figure 5.7. Differences on the frequency of atresia (Kendall's Tau Test) in ovaries from 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not include 
indicate a significant positive or negative association between treatment and degree of 
ovarian atresia (a = 0.05). Ovaries were classified into 3 categories depending on degree 
of atresia. Numbers on top of bars indicate sample sizes (n). 









209 





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had non-detectable levels 
(< 0.001 mg/mL) 



16 






20 






22 



21 



20 



21 



0% 10% 20% 40% 80% 
Effluent Concentration 

Figure 5.8. Mean ± SEM gonadosomatic index (GSI) and vitellogenin concentrations in 
male largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 
20, 40, and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes (n). 
Asterisks indicate differences in relation to controls (ANOVA, Dunnett's multiple 
comparison test; a = 0.05). 






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21 



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20 



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X 

21 



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21 



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0% 10% 20% 40% 80% 
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Figure 5.9. Mean ± SEM 11-ketotestosterone and 1 7p-estradiol concentrations in male 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 or 56 days. Numbers inside histograms indicate sample sizes («). 
Asterisks indicate differences in relation to controls (ANOVA, Dunnett's multiple 
comparison test; a = 0.05). 



211 



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High Spermatogenesis 



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80 



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20 



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Effluent Concentration 

Figure 5.10. Differences on the frequency of testicular development (Kendall's Tau 
Test) in male largemouth bass exposed to different concentrations of paper mill effluent 
(0, 10, 20, 40, and 80%) for 28 and 56 days. 95% Confidence Intervals (CI) that do not 
include indicate a significant positive or negative association between treatment and 
degree of testicular development (a = 0.05). Testes were classified into 2 main categories 
depending on degree of spermatogenesis. Numbers on top of bars indicate sample sizes 
(n). 






212 



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% 10 % 20 % 40 % 80 % 
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Figure 5.11. Mean ± SEM 11-ketotestosterone and 17p-estradiol concentrations in post- 
spawned largemouth bass (n = 10). Fish were exposed to different concentrations of 
paper mill effluent (0, 10, 20, 40, and 80%) for 56 days and then moved to clean water 
ponds for spawning. Asterisks indicate differences in relation to controls (ANOVA, 
Dunnett's multiple comparison test; a = 0.05). 






213 



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0% 10% 20% 40% 80% 
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Figure 5.12. Mean ± SEM of the ratio of 1 7p-estradiol to 1 1-ketotestosterone (E/l 1-KT) 
and of vitellogenin concentrations in post-spawned largemouth bass (n = 10). 
Vitellogenin was not measured in post-spawned males. Fish were exposed to different 
concentrations of paper mill effluent (0, 10, 20, 40, and 80%) for 56 days and then moved 
to clean water ponds for spawning. Asterisks indicate differences in relation to controls 
(ANOVA, Dunnett's multiple comparison test; a = 0.05). 












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28 Days 



56 Days 



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% 10 % 20 % 40 % 80 % 
Effluent Concentration 



Figure 5.13. Relative differences on gonadosomatic index and vitellogenin in female 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 (January) and 56 (February) days, compared to female fish sampled' at ' 
day (December). Asterisks denote significant differences (ANOVA, p < 0.05). 






215 





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56 Days 






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% 10 % 20 % 40 % 80 % 
Effluent Concentration 

Figure 5.14. Relative differences on 11-ketotestosterone and 17p-estradiol in female 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 (January) and 56 (February) days, compared to female fish sampled' at ' 
day (December). Asterisks denote significant differences (ANOVA, p < 0.05). 



216 



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100 

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300 

200 

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% 10 % 20 % 40 % 80 % 

Effluent Concentration 

Figure 5.15. Relative differences on gonadosomatic index and vitellogenin in male 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 (January) and 56 (February) days compared to male fish sampled at day 
(December). Asterisks denote significant differences (ANOVA, p < 0.05). 









217 



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28 Days 



56 Days 



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% 10 % 20 % 40 % 80 % 
Effluent Concentration 



Figure 5.16. Relative differences on 1 1-ketotestosterone and 1 7p-estradiol in male 
largemouth bass exposed to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 28 (January) and 56 (February) days, compared to male fish sampled at day 
(December). Asterisks denote significant differences (ANOVA, p < 0.05). 



218 



10000 



& 7500 

1 

§ 5000 

2500 



100 



£ 75 



W 50 

i 

25 








0% 10% 20% 40% 80% 
Effluent Concentration 



Figure 5.17. Results of the indoor hatchability study showing mean ± SEM of fecundity, 
egg size, percentage of live eggs, and hatchability of eggs spawned by largemouth bass in 
clean fish ponds after an in vivo exposure to different concentrations of paper mill 
effluent (0, 10, 20, 40, and 80%) for 56 days. Eggs were collected from ponds and 
brought indoors for controlled hatchability studies. Hatchability was determined at day 3 
post-hatch. Numbers inside histograms indicate sample sizes (n = number of mats 
collected from ponds and brought indoors). There were no differences with the control 
group (ANOVA, p > 0.05). 



219 



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1 




% 


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& 




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0% 10% 20% 40% 80% 
Effluent Concentration 

Figure 5.18. Results of the indoor hatchability study showing mean ± SEM of yolk 
measurements (yolk width, length, and area) measured from yolk-fry produced by 
largemouth bass in clean fish ponds after an in vivo exposure to different concentrations 
of paper mill effluent (0, 10, 20, 40, and 80%) for 56 days. Eggs were collected from 
ponds and brought indoors for controlled hatchability studies, and measurements taken at 
an average age of 3 days. Numbers inside histograms indicate sample sizes (n = total 
number of fry from which measurements were taken). Asterisks indicate significant 
differences with the control group (ANOVA, Dunnett's multiple comparison test; a - 
0.05). 



220 



Fir Length 



Fry Weight 



1 

20 









1.4 




1.2 


? 


1.0 
0.8 


§ 
1 


0.6 


£ 



Head 

Vertebral Column 

Yolk Sac 



X 2 = 202, p = 0.001 




2,100 



0.4 



0% 10% 20% 40% 80% 
Effluent Concentration 

Figure 5.19. Results of the indoor hatchability study showing mean ± SEM of yolk-fry 
measurements (total length and weight) and percent abnormalities measured from fry 
produced by largemouth bass in clean fish ponds after an in vivo exposure to different 
concentrations of paper mill effluent (0, 10, 20, 40, and 80%) for 56 days. Eggs were 
collected from ponds and brought indoors for controlled hatchability studies, and 
measurements taken at an average age of 3 days. Numbers inside histograms indicate 
sample sizes (n = total number of fry from which measurements were taken). For fry 
length and weight, asterisks indicate significant differences with the control group 
(ANOVA, Dunnett's multiple comparison test; a = 0.05). Differences in the frequency 
distribution of abnormalities were analyzed through a X 2 Test. 



221 



6000 



1 

£ 4000 




0% 10% 20% 40% 80% 
Effluent Concentration 



Figure 5.20. Results from the outdoor hatchability study showing mean ± SEM fry 
produced per spawned female. Fry were produced by largemouth bass in clean fish ponds 
after an in vivo exposure to different concentrations of paper mill effluent (0, 10, 20, 40, 
and 80%) for 56 days. Eggs were left to hatch in ponds, and fry were counted at an 
average age of 13 days. Numbers inside histograms indicate sample sizes (n = total 
number of mats from which fry were collected). Asterisks indicate significant differences 
with the control group (ANOVA, Dunnett's multiple comparison test; a = 0.05). 



222 




£ 



I 3 







Head 

Vertebral Column 

Yolk Sac 



X=19,p = KS. 




3 l 



0% 10% 20% 40% 80% 
Effluent Concentration 



Figure 5.21. Results of the outdoor hatchability study showing mean ± SEM of yolk-fry 
measurements (total length and weight) and percent abnormalities measured from fry 
produced by largemouth bass in clean fish ponds after an in vivo exposure to different 
concentrations of paper mill effluent (0, 10, 20, 40, and 80%) for 56 days. Eggs were left 
to hatch in ponds, and measurements taken at an average age of 14 days. Numbers inside 
histograms indicate sample sizes (n = total number of fry from which measurements were 
taken). For fry length and weight, asterisks indicate significant differences with the 
control group (ANOVA, Dunnett's multiple comparison test; a = 0.05). Differences in 
the frequency distribution of abnormalities were analyzed through a X 2 Test. 



223 



O % O 10 % A 20 % ■ 40 % A 80 % 



S 
& 



130 



120 



110 



100 



J 90 



80 



70 



60 




8 



10 



12 



14 



16 



18 



20 



Age (days) 



Figure 5.22. Results from the outdoor hatchability study showing growth of largemouth 
bass fry. Fry were produced by largemouth bass in clean fish ponds after an in vivo 
exposure to different concentrations of paper mill effluent (0, 10, 20, 40, and 80%) for 56 
days. Eggs were left to hatch in ponds, and fry were collected and measured. Results 
shown only include repeated measurements from fry collected from identifiable mats at 
regular intervals (range of age from 9 to 19 days). There was a significant effect of 
effluent exposure (40 and 80%, black symbols) on fry growth (ANOVA, Dunnett's 
multiple comparison test; a = 0.05). 



CHAPTER 6 

IN VITRO STEROIDOGENESIS BY GONADAL TISSUES FROM FEMALE 

LARGEMOUTH BASS EXPOSED TO PAPER MILL EFFLUENTS AND RESIN 

ACIDS 



Introduction 

Several laboratories, including ours, have demonstrated reductions in circulating 
levels of 17P-estradiol and testosterone in female fish exposed to bleached kraft pulp mill 
effluent (BKME) (Munkittrick et al. 1992a, 1994, McMaster et al. 1996b). Measures of 
in vivo steroid plasma concentrations indicate overall effects on circulating steroid 
hormones but do not provide information as to the site or sites at which pollutants may 
exert their effect(s). Decreased plasma steroids in effluent exposed fish could then be a 
result of inhibition of hypothalamic gonadotropin-releasing hormone (GnRH) or pituitary 
gonadotropin (GtH); decreased gonadal steroid precursors; decreased activity of specific 
enzymes involved in sex steroid biosynthetic pathways; or increased rate of hepatic 
catabolism and excretion of sex steroids (Kime 1995). In vitro incubations of gonadal 
tissue thus, can provide valuable information regarding mechanistic effects of paper mill 
effluents. 

Although several reports have documented reproductive endocrine alterations in 
fish exposed to BKME, few studies have attempted to identify the chemical(s) 
responsible for such changes. Several types of resin acids (including abietic (AA) and 
dehydroabietic acids (DHAA)) are found at high concentrations in the resin extracts of 



224 



225 

conifer trees, and thus are present in significant quantities in paper mill effluents. 
Exposure of fish to resin acids has been reported to cause impair liver function (Nikinmaa 
and Oikari 1982), hemolysis (Bushnell et al. 1985) and neurological dysfunction (Zheng 
and Nicholson 1998). There is also growing evidence that polyunsaturated fatty acids 
(PUFAs) are important regulators of steroid biosynthesis in fish. For example, 
testosterone production in rainbow trout ovarian follicles was inhibited in a dose-related 
manner after exposure to PUFAs (Mercure and Van Der Kraak 1995). Because of the 
chemical similarities between resin acids and PUFAs, it is possible that reproductive 
effects seen in fish exposed to paper mill effluents might be caused by resin acid 
exposure. 

The objectives of this study were to evaluate the effects of acute (7 days) and 
chronic (56 days) BKME exposures on plasma steroid concentrations of largemouth bass 
(Micropterus salmoides). The ability of ovarian follicles from chronically effluent- 
exposed females to secrete hormones in vitro both basally and in response to human 
chorionic gonadotropin (hCG) was also evaluated. In vitro ovarian steroidogenesis was 
further examined after exposure of ovarian follicles to different concentrations of two 
resin acids (abietic acid (AA) and dehydroabietic acid (DHAA)). 

Materials and Methods 

Effluent Characteristics 

The effluent tested in this study comes from a paper mill that has two bleached 
(40% product) and one unbleached line (60% product), which together release an 
estimated 36 million gallons of effluent/day. The bleaching sequences for the bleach line 



226 

are CEHD and CgodioEopHDp (see Chapter 2 for a description of abbreviations). The 
bleaching lines manufacture paper towels and tissue paper, whereas the unbleached line 
produces mainly kraft bag and linerboard. The wood furnish of this mill consists 
typically of 50% softwood (slash, sand, loblolly, pine) and 50% hardwood (gums, tupelo, 
magnolia, water oaks and hickory) species. At the time of this study, effluents received 
secondary treatment, which consisted of both anaerobic followed by aerobic biological 
degradation after a retention period of 40 days. The average (range) concentrations of 
AA and DHAA in the effluent under study are 5.9 (3.8 - 15.5) and 6.6 (3.0 - 16.1) mg/L, 
respectively (calculated from data collected between January and May 1999) (Quinn 
2000). 
In Vivo Exposures 

Experiment 1 . Reproductively active largemouth bass were exposed to five paper 
mill effluent concentrations (0, 10, 20, 40, and 80 %) for a total of 56 days (about 20 
bass/treatment). In order to assess possible effects of short-term effluent exposure, bass 
were also exposed to effluents for 7 days but only for the 0, 40, and 80% concentrations 
(approximately 10 fish/treatment). Fish were held outdoors in five-l,500L plastic, flow- 
through design tanks. In-line digital flow meters (ECOSOL®, Ontario, Canada) were set 
in each tank to control well and effluents inputs and enable appropriate effluent 
concentrations. Fish were fed once a week with commercial fish pellets (Zeigler®). At 
the end of each exposure period, fish were bled for determination of sex steroids using 
radioimmunoassay (RIA). Ovaries were collected for in vitro cultures after 50 days of 
exposure to effluents. 



227 
In Vitro Gonadal Cultures 
Chemicals 

Dehydroabietic (DHAA) and abietic acids (AA) were purchased from Helix 
Biotech (Vancouver, BC, Canada). Human chorionic gonadotropin hormone (hCG), 
minimum essential medium eagle (MEM), and the antibiotics penicillin and streptomycin 
were obtained from Sigma Chemical (St. Louis, MO, USA). Bovine serum albumin 
(BSA) was obtained from Calbiochem Corporation (La Jolla, CA, USA). All other 
solvents and chemicals used in this study were of analytical grade. 
General protocol 

In vitro steroid production was assessed using the following protocol. Follicular 
tissue weighing 115mg was placed in 24-well incubation plates that contained MEM with 
0.1% BSA and 0.01% penicillin and streptomycin. Culture plates were incubated for 48 
hrs in an atmosphere of 4% C0 2 at 26.5°C. Prior to beginning the incubations, half of the 
wells received lOOul of hCG (50 IU/ml, a potent GtH agonist in fish) for a final 
incubation volume of 1.5mL. For each fish, follicles were incubated with and without 
(basal) hCG (x 3 replicates). Negative controls (wells with no tissue and with medium 
with and without hCG) were run with each plate. At the end of the incubations, medium 
was collected, centrifuged at 3,000r.p.m. for lOmin, and stored at -80 °C prior to 
measurement of testosterone or 17(3-estradiol by RIA. 
Specific protocols 

Experiment 2. The objective of this experiment was to determine if exposure of 
largemouth females to whole paper mill effluent had direct effects on ovarian biosynthetic 



228 

capacity. On day 50 of in vivo exposure to effluents, 3 females were collected from each 
treatment tank: 0, 10, 20, 40, and 80% paper mill effluent concentrations. Each animal 
was weighed, blood collected for the determination of testosterone and 17P-estradiol 
concentrations by RIA, sacrificed, and returned to the laboratory on ice. In the laboratory, 
gonads were collected, and immediately placed in chilled MEM for measurement of in 
vitro steroid production by RIA. 

Experiment 3 . The objective of this experiment was to examine the effects of two 
resin acids (DHAA and AA) on largemouth bass steroidogenesis in vitro. Following the 
same protocol of in vitro incubations explained earlier, follicles were collected from two 
vitellogenic control females and exposed to five concentrations of DHAA or AA: 
(controls), 50, 100, 500, and 1000 ug/L. Follicles in each treatment were incubated for 48 
hrs with and without hCG and the production of testosterone and 1 7p-estradiol measured. 
Statistical Analyses 

Pairwise comparisons were conducted using a one-way analysis of variance 
(ANOVA) (PROC GLM, SAS Institute 1988) to test whether treatment effluent 
concentration caused significant differences in plasma sex steroid concentrations. If the 
ANOVA showed significant effluent concentration effects, a Dunnett's multiple 
comparison test was used to examine which effluent concentration(s) differed from the 
control group. For the in vitro study, concentrations of sex steroids were averaged for the 
basal and hCG replicates. The percent induction in hormonal production by ovarian 
follicles after stimulation with hCG was analyzed using non-parametric statistics 
(Wilcoxon Test, PROC NPAR1WAY. Statistical significance was assessed at/? < 0.05. 



229 
Results 

Experiment 1 

Despite the short length of exposure, fish of both sexes exposed to 40 and 80% 
BKME for 7 days showed a decline in plasma concentrations of 1 1-ketotestosterone and 
178-estradiol (Figure 6.1). Vitellogenin concentrations in females decreased by 68% but 
only in the 80% effluent group (data not shown). Similarly, female bass chronically 
exposed to BKME for 56 days showed a significant decline in the concentrations of 
testosterone and 173-estradiol but at lower effluent concentrations (10% or higher) in 
relation to controls (Figure 6.2). In addition, declines in sex steroids were accompanied 
by reductions in vitellogenin concentrations (average decline of 68%) in females exposed 
to 20, 40, and 80% BKME (data not shown). 
Experiment 2 

Gonadal tissue was collected at day 50 from some females and incubated for 48 
hrs for determination of steroid production. In vitro production of 173-estradiol was 
significantly reduced in both basal and hCG-induced follicles (Figure 6.3). This 
reduction was most evident in the latter group, where 17P-estradiol concentrations were 
decreased at all exposures. Testosterone production on the other hand, was more variable 
and reduced only in hCG-stimulated follicles collected from females exposed to high 
paper mill effluent concentrations (40 and 80%) (Figure 6.3). In addition, there was a 
significant decline in the ability of follicles to increase testosterone production after 
stimulation with hCG (50 IU/ml) in females exposed to 20, 40, and 80% effluent 
concentrations (Figure 6.4). 



230 
Experiment 3 

Ovarian steroidogenesis after AA and DHAA in vitro exposure is presented in 
Figures 6.5 and 6.6, respectively. Abietic acid had no effect on the production of 17(3- 
estradiol by gonadal tissue, under both basal and hCG-induced conditions. Testosterone 
production was significantly reduced but only at the highest exposure dose (1000 ug/L of 
abietic acid) and after stimulation with hCG (Figure 6.5). There were no differences in 
the in vitro basal and hCG-stimulated production of both sex steroids after exposure to 
DHAA (Figure 6.6). 

Discussion 

Short-term exposure (7 days) tests were conducted as a way to assess their 
potential for predicting effects noted in long-term studies (i.e. 56 days). Results from 
these preliminary tests (only the 40 and 80% effluent concentrations were evaluated) look 
promising because they show similar changes in biochemical markers (declines in sex 
steroids in both sexes and in vitellogenin in females) when compared to long-term 
exposures. These quick endocrine changes, as well as the similar reductions in 17(3- 
estradiol observed in both the in vivo and the in vitro experiments are suggestive of 
chemical(s) acting locally in the gonad. At this time however, reproductive dysfunction 
at the hypothalamic or pituitary levels, as well as on the peripheral metabolism of steroids 
in fish exposed to these effluents cannot be ruled out. 

Rapid declines in steroid production after exposures to about 50% BKME have 
also been reported from caged and laboratory studies using goldfish (Carassius auratus) 
(McMaster et al. 1996a). In the laboratory, although goldfish exposed to BKME for 4 or 



231 

8 days tended to have reduced circulating concentrations of sex steroids, there was a lack 
of treatment differences probably related to a high degree of variation between fish within 
treatments. Field exposures, however, revealed significant reductions in steroid 
production after in vitro incubations of both male and female goldfish gonadal tissue 
(McMaster et al. 1996a). 

The effects of BKME on testosterone concentrations in female largemouth bass 
were not as clear as those observed for 17p-estradiol. Although plasma testosterone was 
decreased in females exposed to all effluent dilutions for 56 days (this hormone was not 
measured in the 7-day experiment), results from the in vitro cultures were more variable 
with both increases and declines for basal and hCG stimulated conditions, respectively. It 
is worth noting that after 50 days of exposure to paper mill effluents, follicles were still 
capable of responding to hCG stimulation by increasing their production of 17|3-estradiol 
in relation to the controls. However, the functional competence of these same follicles 
appeared impaired for testosterone production at high effluent exposures. Since hCG acts 
as a GtH analog stimulating steroid production via GtH receptors, declines in testosterone 
production after hCG stimulation could have been caused by alterations at the level of 
these receptors. However, since follicles remained competent in the production of 170- 
estradiol after hCG stimulated conditions, declines in testosterone production are more 
likely to have been caused by declines in steroid precursors and/or in the activity or levels 
of the different enzymes that participate in the biosynthesis of sex steroids. The steroid 
pathway in fish first involves the conversion of hydroxy cholesterol into pregnenolone by 
a P450 side chain cleavage enzyme (a step considered rate-limiting) (Nagahama 1994), 
and is followed by the involvement of at least another three enzymes that biotransform 






232 

pregnenolone into testosterone (36-hydroxysteroid dehydrogenase, 1 7a-hydroxylase 
Cn,2o-lyase, and 178-hydroxysteroid dehydrogenase, (McMaster et al. 1996b). 
Reductions in the production of testosterone, then, could have been due to alterations in 
any of these steps. Declines in the production of 1 7p-estradiol, on the other hand, could 
involve similar alterations in addition to impairments in concentrations and/or activity of 
aromatase, the enzyme responsible for the conversion of androgens into estrogens. 
Reductions in testosterone and 17p-estradiol production by follicles collected from 
BKME-exposed white suckers were attributed to reduced levels of aromatase during early 
vitellogenic stages, and to disruptions higher in the steroidogenic pathway (downstream 
of pregenenolone formation) later in the reproductive season (McMaster et al. 1995). As 
already mentioned, a number of sites and functions external to the gonad have also been 
shown to be altered after BKME exposure (e.g. altered pituitary function with reduced 
concentrations of GTH-II and altered peripheral steroid metabolism) (Van Der Kraak et 
al. 1992) 

The general absence of effects from the in vitro exposures of follicles to resin 
acids could be due to several factors, the most relevant probably related to concentration 
and length of exposure. The concentrations chosen in the present study were based solely 
on AA and DHAA levels from whole effluents collected during the time of the study, 
which represent concentrations that are between 130 times to 7 times higher than the 
concentrations used in the lowest and highest in vitro exposure groups, respectively. 
There is almost no information regarding resin acid concentration in gonads of fish 
exposed to paper mill effluents. The only study available on this subject reports an 
average concentration of 6 mg/kg in gonads of rainbow trout (Salmo gairdneri) exposed 



233 

to 1.2 mg/L of DHAA for four days, as opposed to 101 and 86 mg/kg in liver and kidney, 
respectively (Oikari et al. 1982). These findings suggest the need for more chronic 
exposures and/or for the use of higher doses ( > 1 mg/kg) of resin acids if significant 
effects are intended under in vitro conditions. It is also possible that in order for these 
acids to exert their effects, they need to be metabolized in vivo to some unknown 
compound (s) which would be absent under in vitro conditions. 

Results from studies on white sucker {Catostomus commersoni) from Jackfish 
Bay indicate that several sites within the pituitary-gonadal-axis are affected after exposure 
to BKME. Fish from exposed sites had significantly lower plasma levels of GtH-II and 
showed depressed responsiveness of sex steroids and 17,208-dihydroxy-4-pregnen-3-one 
(17,208-P, a maturation-inducing steroid) after injections with GnRH (Van Der Kraak et 
al. 1992). BKME-exposed fish also had lower circulating levels of testosterone 
glucoronide, which would be suggestive of altered peripheral steroid metabolism. 
Similarly to what was observed under in vivo conditions, in vitro incubations of ovarian 
follicles collected from BKME-exposed females have also shown reduced production of 
testosterone, 17p-estradiol, and 17,206-P 2 under basal and hCG stimulated conditions 
(Van Der Kraak et al. 1992, McMaster et al. 1995). The similarities between both types 
of study would suggest that reductions in plasma steroid levels in BKME-exposed fish 
from Jackfish Bay are mainly due to alterations in ovarian steroid production. At this 
point, these multiple endocrine effects are difficult to classify as estrogenic or androgenic. 
This is not surprising considering the fact that BKME are complex mixtures capable of 
containing chemicals with simultaneous antiestrogenic, estrogenic, and even 
androgenic/antiandrogenic properties. 



234 

Recently, several reports have implicated (3-sitosterol, a plant sterol, as a possible 
significant factor contributing to the reproductive effects observed in fish exposed to 
paper mill effluents. In goldfish, injection of P-sitosterol causes reductions in plasma 
circulating levels of testosterone, 11-ketotestosterone, and 17(3-estradiol, and decreases in 
vitro gonadal testosterone and pregnenolone production (MacLatchy et al. 1997). 
Antiestrogenic activity of pulp and paper mill black liquor has also been detected using 
mammalian in vitro recombinant receptor/reporter bioassays (Zacharewski et al. 1995). 
This compound can also induce estrogenic effects in fish: it can bind to the rainbow trout 
estrogen receptor and promote expression of the vitellogenin gene in vitro and in vivo 
(Mellanen et al. 1996, Tremblay and Van Der Kraak 1998). Other compounds present in 
paper mill effluents that have been reported to cause reproductive dysfunction in fish 
include phenol and sulfide. Both of these chemicals inhibited the uptake of radiolabeled 
cholesterol into carp (Cyprinus carpio) ovary from the peripheral circulation and its 
ovarian conversion to progesterone and pregnenolone (Mukherjee et al. 1991). 

There is also evidence suggesting that compounds present in paper mill effluents 
are capable of mediating endocrine responses through receptors other than the estrogen 
receptor. Female mosquitofish, Gambusia affinis, inhabiting a stream receiving paper 
mill effluents in Florida were strongly masculinized showing both physical secondary 
sexual characteristics (fully developed gonopodium) and reproductive behavior of males 
(Howell et al. 1980). More recently, masculinization of female fish has been identified 
from an additional two species (least killifish, Heterandria formosa and sailfin molly, 
Poecilia latipinna) collected from Rice Creek, the stream receiving the effluents 
discharged by the Palatka mill (Bortone and Cody 1999). Masculinization of female fish 



235 

has been attributed to the action of androgenic hormones that result from the 
biotransformation of plant sterols (and also cholesterol and stigmasterol) by bacteria such 
as Mycobacterium (Howell and Denton 1989). The concentration of P-sitosterol in the 
effluent under study (average of 292 ug/L, range 200 - 549 ug/L) falls within the range of 
concentrations known to affect fish reproduction. It is clear that additional studies are 
required for a better understanding of the role of plant sterols and resin acids on the 
reproductive physiology of largemouth bass. 












236 



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Asterisks indicate differences in relation to controls (ANOVA, Dunnett's multiple 
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female largemouth bass exposed to different concentrations of paper mill effluents for 56 
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indicate significant difference in relation to controls (ANOVA, Dunnett's multiple 
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238 



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follicles from largemouth bass exposed in vivo to different concentrations of paper mill 
effluents (0, 10, 20, 40, and 80%) for 50 days (experiment 2). Values graphed are pooled 
means obtained from three fish/treatment, each incubated separately and in triplicate with 
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ovarian follicles with 50 IU/ml hCG in relation to basal production. Follicles were 
collected from largemouth bass exposed in vivo to different concentrations of paper mill 
effluents (0, 10, 20, 40, and 80%) for 50 days (experiment 2). Values graphed are pooled 
means obtained from three fish/treatment. Effluent exposure of 20% or higher caused a 
significant decrease in the ability of follicles to produce testosterone after hCG 
stimulation (Wilcoxon Test, X 2 = 14.6, p = 0.006). 



240 



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from largemouth bass exposed in vitro to different concentrations of abietic acid (0, 50, 
100, 500, and 1000 ug/L) for 48 hours (experiment 3). Values graphed are pooled means 
obtained from two clean females, each incubated separately and in triplicate with and 
without hCG. Asterisks indicate significant difference in relation to controls (ANOVA, 
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241 



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from largemouth bass exposed in vitro to different concentrations of dehydroabietic acid 
(0, 50, 100, 500, and 1000 ug/L) for 48 hours (experiment 3). Values graphed are pooled 
means obtained from two clean females, each incubated separately and in triplicate with 
and without hCG. There were no significant differences in relation to controls (ANOVA, 
Dunnett's multiple comparison test; a = 0.05). 












CHAPTER 7 
GENERAL CONCLUSIONS, ECOLOGICAL SIGNIFICANCE, AND FUTURE 

RESEARCH NEEDS 



General Conclusions 

Over the course of three years, the potential effects of bleached kraft paper mill 
effluents (BKME) on health and reproduction of largemouth bass (Micropterus 
salmoides) were examined through a multi-tiered approach that consisted of field 
investigations as well as paired in vivo and in vitro laboratory studies. The following 
section outlines the major findings from these studies. 
Field Studies 

Field studies were conducted in two phases (1996/97 and 1998) with the objective 
of evaluating several physiological responses of free-ranging populations of largemouth 
bass in relation to BKME exposure. For these studies, bass were collected from effluent 
exposed sites and reference sites (usually located upstream from the effluent discharge), 
and responses compared across sites. Exposure and effects due to BKME were assessed 
using bioindicators that covered several levels of biological organization, ranging from 
suborganismal-level responses, such as biochemical, physiological, and histological, to 
organism and population-levels responses. 

In general, results from these field studies revealed that lower and potentially 
sensitive levels of biological organization, biochemical and physiological (such as 
concentrations of sex steroids and vitellogenin and induction of biotransformation 

242 



243 

enzymes), were altered in largemouth bass from streams closest to the mill discharge 
(Rice Creek and Palatka sites). Reproductive alterations were also observed in fish 
sampled at considerable distances from the mill (Green Cove and Julington Creek located 
at 40 and 55km from the mill, respectively), which would suggest exposure to chemicals 
other than BKME. Changes in sex steroids and vitellogenin, however, were not 
necessarily related to impacts at higher and less sensitive levels of organization such as 
organ (gonad weights), organism (fecundities), and population-levels (age distributions). 
Health assessment evaluations showed that some endpoints were altered in bass collected 
from exposed streams; however, these changes fell within normal physiological ranges 
and were probably not associated with detrimental health effects. Finally, size of stream 
(mainstream vs. tributary) and timing of sampling during the reproductive season were 
important factors influencing many of the endpoints measured. 
In Vivo Studies 

Laboratory studies that involved exposures of largemouth bass to different 
concentrations of BKME (10, 20, 40, and 80%) for up to 56 days were conducted during 
the reproductive seasons of 1998 and 1999. Several reproductive and health endpoints 
were measured in effluent-exposed fish and compared to controls. 

Exposure to BKME in the laboratory was assessed through the measurement of 
resin acids in bile, and it was concluded that this could serve as a suitable biomarker of 
short-term exposure to paper mill effluents in populations of largemouth bass. In contrast 
to what was observed under field conditions, in vivo exposures resulted in biochemical- 
level changes (decline in sex steroids and vitellogenin) that were usually translated into 
tissue/organ-level responses (declines in GSIs and retardation of gonadal development). 



244 

The majority of these responses were observed after exposures to at least 20% BKME 
concentrations. It was also observed that endocrine alterations in females were reversible 
after a depuration period of about a month, but were persistent in males. In addition, 
laboratory studies demonstrated that sex and length of exposure were important factors 
influencing many of the observed responses, with an overall higher susceptibility of 
females with respect to males and a general increase in responses in fish exposed to 
effluents for 56 days in relation to 28 days. 

From the 1999 spawning study, declines in sex steroids, vitellogenin and GSIs did 
not result in lower fecundities (measured as number of eggs spawned) and egg sizes or in 
decreased hatchabilities. Later evaluations of fry numbers, however, revealed significant 
negative effects of effluent exposure on survivorship, with a threshold effluent 
concentration of 10%. It was hypothesized that the decline in fry numbers could have 
been caused by an increased frequency of deformities coupled with alterations in growth. 
These changes in turn, could have resulted from acute toxicity to embryos after 
translocation of persistent organic compounds from the mother to the developing embryo 
and/or from chronic failure of parental reproductive systems after almost two months of 
effluent exposures. It is also possible that changes in fry relate to alterations in the 
"quality" of the yolk that was being deposited in the developing oocyte during the course 
of the in vivo exposures. Since vitellogenin serves as an important carrier molecule for 
essential nutrients (metals, ions, vitamins, lipids, and hormones) into the developing 
ovary, the decline in the production of vitellogenin by livers of BKME-exposed females 
could have resulted in concomitant decreases in the amounts and/or types of essential 



245 

nutrients that were being mobilized into the developing embryo, negatively affecting 
normal fry development. 
In Vitro Studies 

Gonads were collected from females that had been exposed to effluents for 50 
days, and sex steroid production measured in vitro. In addition, since resin acids are 
known to be present at high concentrations in the Palatka effluent, preliminary attempts 
were directed towards evaluating the potential effects of such chemicals on the 
steroidogenic capacity of isolated ovarian follicles. 

There was a dose-dependant decline in the production of 17P-estradiol by follicles 
collected from BKME-exposed females. This finding suggested the direct action of 
chemical(s) at the gonad level, since these declines paralleled changes in plasma 17P- 
estradiol observed in females during the in vivo studies. This hypothesis was further 
supported after short-term in vivo exposures (7 days) resulted in similar reductions in sex 
steroids and vitellogenin. In addition, there were no dose-response changes associated 
with resin acid exposures. These results would suggest the action of chemicals other than 
resin acids (e.g. phytosterols or chlorinated organics) as possible causative agents of the 
reproductive alterations observed in BKME-exposed largemouth bass. 

Ecological Significance 

One of the main objectives of controlled laboratory studies is to estimate threshold 
concentrations capable of causing specific alterations. Once effluent concentrations in 
the receiving streams are known, the next step involves careful extrapolation of results 
obtained under controlled conditions for use in evaluations of impacts in the field. It is 



246 



important to keep in mind, however, that these extrapolations are subject to many 
limitations, including uncertainties regarding impacts at higher levels of biological 
organization (population, community, and ecosystem) and difficulties in establishing 
chemical exposures in free-ranging fish. In this respect, the results from our laboratory 
studies suggest negative effects of BKME on fry growth and survival and on plasma sex 
steroid and vitellogenin concentrations at threshold levels of 10 and 20%, respectively. 
These threshold values fall within the 60% average yearly concentration of effluent that 
exists in the stream near the point of discharge (Rice Creek), and thus would suggest 
probable population-level effects in largemouth bass inhabiting this small stream. Our 
laboratory studies would also predict that endocrine alterations are less likely to occur in 
bass inhabiting streams further away from the mill, such as at the confluence of Rice 
Creek with the St. Johns River (Palatka site) where effluents are estimated to be under 
10%. Alterations in fry growth and survival, however, occurred after exposures to only 
10% BKME. Since the lowest effluent concentration tested in this study was 10%, it 
remains unknown if bass from the Palatka site could potentially be affected by such an 
exposure as well. In addition, because many reproductive responses observed in BKME- 
exposed bass were intensified with length of dosing, exposures of over 56 days could 
result in increasingly lower threshold concentrations, leading to population-level effects 
in low-level effluent streams. 

As already mentioned, an important limitation of field studies has to do with the 
inability to accurately assess chemical exposures. From the laboratory studies, most of 
the responses occurred after 56 days of effluent exposures. Because largemouth bass are 
a mobile species, almost two months of continuous effluent exposure may not be an 



247 

ecorelevant scenario. Insufficient effluent exposure in free-ranging largemouth bass 
could explain the lack of association between decreased hormone and vitellogenin 
concentrations and obvious reproductive impairment (such as alterations in gonad 
weights and gonad developments) observed in our field studies. It is also important to 
mention that, even though results obtained from the in vivo experiments indicate declines 
in sex steroids and vitellogenin after exposures to 20% BKME, effects on gonad weights 
and histology are not seen unless fish are exposed to 40 and 80% effluents. Although 
effluent concentrations in Rice Creek can reach over 90% during periods of low-water 
flow, the scarcity of bass in this stream would indicate absence of adequate prey and/or 
nesting substrate, thus making this area unsuitable for long-term residency. As discussed 
in the next section, future studies on the environmental effects of paper mill effluents 
should incorporate clear biomarkers of exposure, intensive chemical analytical 
characterization, and should consider the use of less mobile species as additional study 
models. 

Future Research Needs 

Although our research on the effects of BKME on largemouth bass constitutes one 
of the most thorough attempts ever conducted in the United States on this subject, there 
are still many unanswered questions. The following is a list of some suggestions that 
should help improve our knowledge and understanding on the potential environmental 
effects of pulp and paper mill effluents. 



248 

Additional Field Studies 

There is a real need for more environmental studies that focus, not only on 
biochemical and organism-level effects associated with paper mill effluent exposures, but 
that include measurement of population, community, and ecosystem-level responses. 
Although these changes are difficult to measure, and may require many years of field 
studies, they are absolutely essential if our goal is to predict environmental impacts. 
Prediction of biological impacts due to effluents, however, will not be possible without 
the development of biomarkers of exposure. This is of particular importance when 
working with mobile species, such as largemouth bass. Our studies have demonstrated 
that induction of EROD activity (the preferred biomarker of BKME exposure) may not be 
as useful when measured during the reproductive season, and thus alternative methods 
(such as measurement of BKME-specific chemicals in tissues) need to be developed. 
Since paired measurements of biological responses and of exposure to environmental 
contaminants are needed for meaningful data interpretation, future field studies should 
incorporate thorough tissue chemical characterization of fish from effluent-exposed and 
reference sites. 
Mesocosms Studies 

For our laboratory exposures, bass were held in relatively small tanks and were 
fed commercial pellets. Because of the limited tank space, they had to be moved to ponds 
for spawning which precluded us from measuring reproductive success endpoints while 
being dosed with effluents. Exposure of bass to environmental relevant effluent 
concentrations through the use of ponds (mesocosms approach) would allow the 
evaluation of effects on early life stages (eggs and fry) after continuos effluent exposure 






249 



of adults. In addition, if properly maintained with sources of natural prey, ponds could 

hold offspring for prolonged periods of time for the conduction of multigenerational 

studies. 

Evaluate Effects on Other Aquatic Organisms 

Since several studies have demonstrated differences in susceptibility to the effects 
of BKME exposure, it would be of great value to measure responses in other fish species. 
Some factors to consider before choosing other fish models include: knowledge of the 
organism's biochemistry and physiology, as well as on basic population parameters; 
availability; size and ease of sampling; trophic level and degree of movement; and 
ecosystem/societal importance. Effects of effluents on aquatic invertebrates should also 
be considered, since they offer the advantages of having smaller home ranges and are 
better suited to withstand caged studies in relation to fish. In this respect, ongoing studies 
on the effects of BKME on freshwater mussels have shown that these organisms could 
serve as a good indicator species for future environmental studies. 
Evaluate Biological Effects of Mill Improvements 

The Palatka paper mill plant has been in operation for over 50 years. Presently, 
this mill is implementing a series of important renovations necessary to comply with the 
US EPA cluster rule promulgated in 1998. Some of these changes include the use of 
chlorine dioxide bleaching instead of elemental chlorine and of oxygen and hydrogen 
peroxide bleaching instead of sodium hypochlorite. Improvements in secondary 
treatment of effluents are also underway. It is expected that these changes will result in 
the production of a "cleaner" and less toxic effluent. Results from subsequent studies at 






250 

this site will provide insight on the efficacy of these changes, providing useful 
information to both the industry and scientific communities. 
Mechanistic Studies 

Paper mill effluents are complex mixtures that may contain several hundred 
different compounds. Their chemical composition is also very dynamic, and likely to 
change depending on factors such as species of trees that are being pulped, and 
differences on cooking and bleaching techniques. It is probably because of this that still 
today, very little is known about the chemical(s) responsible for the altered reproductive 
functions observed in fish exposed to these effluents. Less is known about chemical 
interactions and modes of action. This area of research should be given first priority 
during the next few years. 









REFERENCES 

Adams, S.M., Crumby, W.D., Greeley, M.S., Shugart, L.R., and Saylor, C.F. 1992. 

Responses of fish populations and communities to pulp mill effluents: A holistic 
assessment. Ecotoxicology and Environmental Safety 24: 347-360. 

Adams, S.M., and McLean, R.B. 1985. Estimation of largemouth bass, Micropterus 
salmoides Lacepede, growth using the liver somatic index and physiological 
variables. Journal of Fish Biology 26: 1 1 1-126. 

Adams, S.M., Shepard, K.L., Greeley, M.S., Jimenez, B.D., Ryon, M.G., Shugart, L.R., 
and McCarthy, J.F. 1989. The use of bioindicators for assessing the effects of 
pollutant stress on fish. Marine Environmental Research 28: 459-464. 

Ahokas, J.T., Holdway, D.A., Brennan, S.E., Goudey, R.W., and Bibrowska, H.B. 1994. 
MFO activity in carp (Cyprinus carpio) exposed to treated pulp and paper mill 
effluent in Lake Coleman, Victoria, Australia, in relation to AOX, EOX, and 
muscle PCDD/PCDF. Environmental Toxicology and Chemistry 13: 41-50. 

Anderson, M.J., Miller, M.R., and Hinton, D.E. 1996. In vitro modulation of 17-B- 

estradiol-induced vitellogenin synthesis: Effects of cytochrome P4501A1 inducing 
compounds on rainbow trout (Oncorhynchus mykiss) liver cells. Aquatic 
Toxicology 34: 327-350. 

o 

Andersson, T., Forlin, L, Hardig, J., and Larsson, A. 1988. Biochemical and 

physiological disturbances in fish inhabiting coastal waters polluted with bleached 
kraft mill effluents. Marine Environmental Research 24: 233-236. 

Axelsson, B., and Norrgren, L. 1991. Parasite frequency and liver anomalies in three- 
spined stickleback, Gasterosteus aculeatus (L.), after long-term expoure to pulp 
mill effluents in marine mesocosms. Archives of Environmental Contamination 
and Toxicology 21: 505-513. 

Bailey, R.M., and Hubbs, C.L. 1949. The black basses (Micropterus) of Florida, with 
description of a new species. University of Michigan Museum of Zoology, 
Occasional Papers 516: 1-40. 

Bankey, LA., Van Veld, P.A., Borton, D.L., LaFleur, L, and Stegeman, J.J. 1994. 

Responses of cytochrome P4501A in freshwater fish exposed to bleached kraft 



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BIOGRAPHICAL SKETCH 

Maria Soledad Sepulveda was born in Santiago, Chile, on April 6, 1967. From 
1972 to 1984, she attended elementary and high school at the Saint John's Villa 
Academy, Santiago. In 1985, Maria began her college education at the Universidad de 
Chile in Santiago. She was awarded a bachelor of science degree in veterinary sciences 
in 1990 and a doctor of veterinary medicine degree in 1991. In the fall of 1997, she 
received the degree of master of science in the Department of Wildlife Ecology and 
Conservation at the University of Florida. Her master's work was on the effects of 
mercury on the health and survival of great egrets in the Everglades. That same year 
Maria began working on the effects of paper mill effluents on the health and reproduction 
of largemouth bass. She was officially accepted as a Ph.D. student in the Department of 
Physiological Sciences, College of Veterinary Medicine, University of Florida, in the 
spring of 1998, and as a Ph.D. candidate in the summer of 1999. Maria received her 
doctoral degree in August 2000. 



268 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




ge^, 



Tirn^nh^SfTiross, Chair 
Associate Scientist of Physiological 
Sciences 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Stephen M. Roberts, Cochair 
Professor of Physiological Sciences 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 

~\k ^J^xfeuH — 

Trenton R. Schoeb 
Professor of Pathobiology 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Evan P. Gallagher 
Assistant Professor of Physiological 
Sciences 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 

Nancy D. Denslow 
Associate Scientist of Biochemistry & 
Molecular Biology 



This dissertation was submitted to the Graduate Faculty of the College of 
Veterinary Medicine and to the Graduate School and was accepted as partial fulfillment 
of the requirements for the degree of Doctor of Philosophy. 

August 2000 




Dean, College of Veterinary Medicine 



Dean, Graduate School 















UNIVERSITY OF FLORIDA 

IIIIIIIIIIIP 

3 1262 08554 3543